NRLF 


IMMUNITY 


METHODS  OF  DIAGNOSIS  AND  THERAPY 

AND 

THEIR  PRACTICAL  APPLICATION 


CITRON 


GARBAT 


I  M  M  UN  IT  Y 

METHODS  OF  DIAGNOSIS  AND  THERAPY 

AND 

THEIR  PRACTICAL  APPLICATION 


BY 

DR.  JULIUS  CITRON^ 

ASSISTANT  AT  THE  UNIVERSITY   CLINIC   OF   BERLIN,  II   MEDICAL  DIVISION 


TRANSLATED  FROM  THE  GERMAN  AND  EDITED 


BY 

A.    L.   GARBAT,   M.   D. 

ASSISTANT  PATHOLOGIST,   GERMAN   HOSPITAL,   NEW  YORK 


27  ILLUSTRATIONS 
'*    '  ^          2  COLORED  PLATES  AND  8  CHARTS 


PHILADELPHIA 

P.   BLAKISTON'S  SON  &   CO. 

1012  WALNUT  STREET; 
1912 


COPYRIGHT,  1912,  BY  P.  BLAKISTON'S  SON   &  Co. 


;     5      Printed  by 
'..'The  Maple  Press 
York,  Pa. 


TO 
PROFESSOR  FRIEDRICH  KRAUS 

AS   EVIDENCE   OF   DUE  HONOR  AND  THANKFULNESS 
THIS   BOOK   IS   DEDICATED 

BY  THE   AUTHOR 

ON   THE   OCCASION   OF  THE   OPENING   OF  THE   NEW 
II   MEDICAL    DIVISION 


6289 


PREFACE  TO  THE  GERMAN  EDITION. 


This  book  is  to  serve  a  purely  practical  purpose.  The  methods  of 
serum  diagnosis,  on  account  of  their  growing  clinical  significance,  are 
constantly  stimulating  greater  interest  in  all  branches  of  medical  science. 
While  giving  instruction  in  this  subject,  I  realized  that  it  would  be  of  great 
help  to  both  the  medical  student  and  physician  if  they  possessed  a  short 
text-book  which  would  review  in  a  purely  critical  form  the  various  methods 
of  immunity  diagnosis,  especially  those  relating  to  tuberculosis  and  syphilis. 

The  two  systems  of  Kolle  and  Wassermann,  and  R.  Kraus  and  Levaditi 
are  doubtless  the  standards  on  the  subject  in  German  medical  literature. 
On  account  of  their  size  and  price,  however,  these  volumes  come  to  be 
sought  only  by  the  specialist. 

It  was  therefore  my  aim  in  this  book  to  so  present  the  subject  of  immu- 
nity that  the  general  medical  man,  who  is  even  slightly  acquainted  with 
laboratory  work,  can  learn  the  details  of  the  various  reactions  and  their 
significance.  In  selecting  the  different  methods,  I  have  taken  up  those 
which  are  used  in  the  clinic  for  diagnostic,  therapeutic,  or  prophylactic 
purposes.  In  addition  there  are  herein  included  certain  fundamental 
considerations  of  questions  on  immunity  which  for  the  present  are  only  of 
theoretical  interest,  but  which  owing  to  the  rapid  development  of  the  subject, 
may  soon  become  of  practical  importance. 

I  have  endeavored  especially  to  place  before  the  reader  a  critical  review 
of  the  results  of  the  various  methods.  In  the  description  of  technical  details, 
the  original  articles  of  the  author  have  been  selected;  modifications  having 
been  considered,  only  provided  they  exhibit  distinct  advantages  over  the 
original  method. 

I  here  wish  to  express  my  thanks  to  my  teacher  Prof.  Wassermann, 
under  whose  guidance  and  stimulus  I  gained  my  laboratory  experience;  also 
to  my  chief  Prof.  Kraus  whose  clinical  genius  proved  to  me  the  practical 
importance  that  this  subject  of  immunity  commands. 

To  the  publishers  as  well,  whose  kind  cooperation  in  all  my  plans  as 
regards  publication  and  illustration,  greatly  simplified  my  work,  I  extend 
my  heartiest  appreciation. 

JULIUS  CITRON. 


Vll 


NOTE  BY  THE  AMERICAN  EDITOR. 


The  study  of  "  Immunity,"  once  of  merely  theoretical  interest  and 
purely  scientific  importance,  is  to-day  no  longer  such.  A  realm  of  practical 
considerations,  considerations  which  are  constantly  coming  up  and  enlisting 
the  attention  of  the  busy  practitioner  have  little  by  little  supplanted  those 
phenomena  at  one  time  vaguely  understood  and  mostly  taken  for  granted. 
Gradually  have  the  uncertainties  so  long  dominating  and  obscuring  an 
intelligent  comprehension  of  the  subject  been  cleared  away;  mistakes 
explained;  and  hypotheses  re-established  as  proven  facts.  The  methods 
employed  for  the  necessary  investigations  have  naturally  improved  and 
increased  with  such  extreme  rapidity  that  a  severe  task  presents  itself  to  one 
who  desires  to  separate  the  more  from  the  less  valuable  ones.  It  was 
therefore  with  extreme  satisfaction  that  I  greeted  the  opportunity  of  bring- 
ing out  an  English  edition  of  this  working  hand-book  on  the  various,  but 
most  essential  methods  used  in  the  applications  of  "Immunity."  The 
author  of  this  volume  has,  by  his  exhaustive  research  and  extensive  practical 
experience  as  a  teacher,  treated  his  field  with  such  fulness  and  preciseness 
of  detail  that  it  is  of  value  not  only  to  the  laboratory  student,  but  also  to  the 
clinician. 

Its  already  favorable  reception  in  Germany  will,  it  is  hoped,  be  extended 
to  it  in  America,  especially  by  those  whose  lack  of  familiarity  with  the  Ger- 
man language  has  kept  this  work  beyond  their  reach. 

The  chapter  on  vaccines  has  been  slightly  revised  and  elaborated  to 
conform  more  closely  with  the  most  recently  advocated  methods  of  Sir  A.  E. 
Wright,  to  whom  the  editor  is  indebted  for  his  experience.  Otherwise 
there  has  been  no  need  to  alter  the  original  text,  with  the  exception  that 
here  and  there  some  features  which  may  be  of  special  interest  to  the  English 
reading  public,  have  been  inserted. 

I  wish  in  the  present  connection  to  express  my  deep  thanks  to  my  teacher 
Dr.  Citron  for  offering  me  the  privilege  of  this  undertaking,  and  to  the 
publishers,  Messrs.  Blakiston  &  Co.,  without  whose  hearty  cooperation 
this  would  have  been  impossible,  my  sincere  appreciation. 

A.  L.  GARBAT. 


IX 


TABLE  OF  CONTENTS. 


CHAPTER  I. 

PAGE. 

INTRODUCTION i 

Definitions  of  immunity  and  antibody.  The  law  of  specificity.  The  necessity 
of  control  tests. 

CHAPTER  II. 

LABORATORY  EQUIPMENT 7 

General  technique.  (Technique  of  injection.  The  methods  of  obtaining  and  pre- 
serving serum.  Bacterial  filtration.  Dilutions.  Measurement  of  small 
amounts  of  bacteria.) 

CHAPTER  III. 

ACTIVE  IMMUNITY 21 

Immunization  with  living  and  dead  virus.  (Vaccination  against  small-pox;  anti- 
rabic  vaccination;  antityphoid  inoculation.) 

CHAPTER  IV. 

ACTIVE  IMMUNITY 34 

Immunization  with  bacterial  extracts.     Aggressin  experiments. 

CHAPTER  V. 
TUBERCULIN  DIAGNOSIS 43 

Koch's  method;  cutaneous  reaction;  Moro's  ointment  reaction;  ophthalmo- 
reaction;  the  specificity  of  the  tuberculin  reactions. 

CHAPTER  VI. 

TUBERCULIN  THERAPY    . 55 

The  technique  of  the  tuberculin  therapy;  old  tuberculin;  new  tuberculin;  bovine 
tuberculin.  Nastin. 

CHAPTER  VII. 

TOXIN  AND  ANTITOXIN 68 

The  serum  therapy  of  diphtheria. 

CHAPTER  VIII. 

TOXIN  AND  ANTITOXIN  (continued) 78 

Definition  of  toxin.  Tetanus  toxin.  Botulism  toxin.  Dysentery  toxin. 
Staphylolysin. 

xi 


Xll  TABLE    OF    CONTENTS. 

CHAPTER  IX. 

PAGE. 

THE  TOXINS  OF  THE  HIGHER  PLANTS  AND  ANIMALS  AND  THEIR  ANTIBODIES.    FER- 
MENTS AND  ANTIFERMENTS     87 

Hay  fever.     Snake  poison.     Paroxysmal  hemoglobinuria. 

CHAPTER  X. 

AGGLUTINATION 97 

Macroscopic  test,  microscopic  test.     Group  agglutination. 

CHAPTER  XI. 

PRECIPITINS 108 

Bacterial  precipitation.     Proteid  precipitation. 

CHAPTER  XII. 

BACTERIOLYSINS  AND  HEMOLYSINS  (CYTOLYSINS) 118 

Technique  of  bacteriolytic  tests.  Pfeiffer's  phenomenon.  Bactericidal  test 
by  plate  method  of  Neisser  and  Wechsberg.  Hemolysins.  Cytolysins. 

CHAPTER  XIII. 

METHOD  OF  COMPLEMENT  FIXATION 139 

Principles  of  this  method.  Antituberculin.  Ehrlich's  side-chain  theory. 
Serum  diagnosis  of  syphilis  and  diseases  caused  by  animal  parasites. 

CHAPTER  XIV. 

TECHNIQUE  OF  THE  COMPLEMENT  FIXATION  METHOD 155 

The  original  method  of  Bordet-Gengou,  Wassermann-Bruck's  modification. 
The  technique  of  the  serum  diagnosis  of  syphilis.  Echinococcus  disease. 
The  differentiation  of  proteids  according  to  Neisser-Sachs. 

CHAPTER  XV. 

PHAGOCYTOSIS.     OPSONINS  AND  BACTERIOTROPINS 174 

Technique  of  opsonic  index  determination  and  of  Wright's  vaccine  treatment. 

CHAPTER  XVI. 

PASSIVE  IMMUNITY.     (SERUM  THERAPY) 192 

Bacteriolytic  sera.     Serum  sickness.     Anaphylaxis.     Special  serum  therapy. 

INDEX 203 


LIST  OF  ILLUSTRATIONS  AND  CHARTS. 


FIG.  PAGE. 

1.  A  room  in  the  laboratory  of  the  Royal  Institute  for  Infectious  Diseases 

(Berlin)      7 

2.  Standard  for  measuring  the  size  of  platinum  loops  (Czaplewski) 8 

3.  Intravenous  inoculation  (after  Uhlenhuth) 9 

4.  Intraperitoneal  inoculation  (after  Uhlenhuth) n 

5.  Removal  of  peritoneal  exudate  in  Friedberger's  position  (original) 12 

6.  Veno-puncture  (original) 13 

7.  Wet-cup  method  for  obtaining  blood  (original) 14 

8.  Test-tube  for  preservation  of  serum  (original) 15 

9.  Pukal  filter 16 

10.  Filtration  through  pukal  filter 16 

11.  Reichel  filter 17 

12.  Lilliputian  filter 17 

13.  V.  Pirquet's  tuberculin  test  (original) 48 

14.  Ophthalmodiagnosticum  for  tuberculosis  (original) 49 

15-16.  Diagram  for  the  complement  fixation  reaction 141 

17.  Diagram  for  the  complement  fixation  in  syphilis 151 

18-20.  Technique  for  the  determination  of  the  opsonic  index  according  to  Wright .    .  181 

21-22.  Technique  for  the  determination  of  the  opsonic  index  according  to  Wright .    .  182 

23-24.  Technique  for  the  determination  of  the  opsonic  index  according  to  Wright .    .  183 
25-26.  Technique  for  the  determination  of  the  opsonic  index  according  to 

Wright 184-185 

27.  Phagocytosis  of  tubercle  bacilli      186 

CHART 

1.  Example  of  a  diagnostic  tuberculin  reaction   .    .- 46 

2.  Example  of  hypersusceptibility  by  diminution  in  the  tuberculin  dose     ...  60 

3.  Marked  increase  of  weight  in  a  tuberculous  individual  in  spite  of  continued 

fever 62 

4.  Treatment  with  S.  B.  E.,  almost  without  reaction.     Immunization  against 

B.  E 65 

5.  Opsonic  curve  after  a  small  dose  of  staphylococcus  vaccine 178 

6.  Opsonic  curve  during  treatment  with  new  tuberculin 179 

7.  Increase  in  the  opsonic  index  for  gonococci  by  Bier's  hyperemia 180 

8.  Auto-inoculation  with  tuberculin  after  physical  examination  and  massage.    .  180 


xill 


IMMUNITY. 


CHAPTER  I. 

INTRODUCTION. — DEFINITIONS  OF  IMMUNITY  AND  ANTIBODY. — THE  LAW 
OF  SPECIFICITY. — THE  NECESSITY  OF  CONTROL  TESTS. 

The  diagnosis  of  infectious  diseases  can  be  approached  in  several  ways. 
In  addition  to  the  aid  obtained  from  clinical  signs  such  as  the  course  of  the 
temperature,  the  changes  in  the  various  organs,  the  exanthemata,  etc.,  the 
finding  of  the  specific  etiological  agent  of  the  disease,  or  the  specific  anti- 
bodies developed  by  the  reaction  of  the  organism  are  of  equal  or  even 
greater  importance.  The  course  of  an  infection  depends  not  only  upon  the 
nature,  the  number,  and  the  virulence  of  the  infecting  agents,  but  also  upon 
the  behavior  of  the  infected  body.  One  must  consider  a  disease  as  the  result 
of  the  interaction  of  both  of  these  factors  without  necessarily  being  able  to  attrib- 
ute the  various  symptoms  to  either  the  one  or  the  other.  Although  the  general 
reaction  of  the  organism  is  varied,  it  can  nevertheless  be  shown  that  in 
spite  of  even  individual  differences,  the  characteristic  bacteria  and  their 
products  bring  about  a  distinct  symptom-complex  which  is  usually  con- 
comitant with  a  significant  defence  on  the  part  of  the  organism.  The  means 
which  the  body  employs  in  this  protection  are  cellular  and  humoral  in  nature. 
In  fact,  there  is  a  group  of  infectious  diseases  in  which  the  cellular  reaction 
predominates,  and  another  in  which  humoral  changes  are  pre-eminent; 
and  between  these  extremes  are  various  intermediate  forms.  Thus  the 
constantly  changing  picture  of  tuberculosis  always  shows  the  tubercle  as  its 
typical  product  of  cellular  reaction;  similarly  leprosy  and  syphilis  have 
their  peculiar  cellular  changes.  More  difficult,  however,  to  recognize  by 
the  unaided  eye  or  even  the  microscope  are  the  finer  biological  alterations 
which  take  place  in  the  body  fluids  during  the  course  of  infectious  diseases. 
Here,  special  methods  are  necessary  to  detect  and  differentiate  the  various 
humoral  changes  which  occur  for  the  main  part,  in  the  blood  serum.  As  is 
known  at  present,  the  humoral  as  well  as  the  cellular  immunity  reactions 
are  not  limited  to  infectious  diseases,  but  also  'express  normal  physiological 
and  pathological  conditions.  With  the  conception  of  Ehrlich's  side-chain 
theory  the  bridge  of  understanding  for  the  humoral  reaction  was  built,  and 


2  INTRODUCTION. 

it  at  once  became  evident  how  the  physiological  phenomena  of  nutrition 
and  production  of  energy  are  identical  in  their  nature  with  processes  which 
under  pathological  circumstances  lead  to  the  formation  of  anti-infectious 
bodies.  In  an  analogous  and  no  less  ingenious  manner,  Metschnikoff  has 
shown  that  the  same  cell  group  of  mesenchymal  origin  which  the  organism 
stations  against  bacterial  invasion  has  physiological  and  physio-patho- 
logical functions  to  fulfill  in  the  whole  animal  scale.  In  the  lower  animals, 
these  cells  aid  in  the  metamorphosis  of  the  body  structure,  thus  leading  to 
the  disappearance  of  entire  organs.  In  the  female,  they  aid  in  the  involu- 
tion of  the  uterus  after  labor,  while  in  the  aged,  they  destroy  the  nerve  cells 
in  the  senile  atrophied  nerve  centers  or  finally  as  chromophages  turn  the 
hair  gray.  The  border-line  between  the  physiological  and  pathological  status  is 
biologically  not  sharply  demarcated.  It  is  one  single  chain  of  manifestations 
which  possess  numerous  transitional  phases.  As  the  methods  of  serum 
diagnosis  can  prove  reactions  much  finer  even  than  those  accomplished  by 
chemistry,  their  application  has  not  been  limited  to  the  chapter  on  infectious 
diseases. 

By  their  means  also,  proteids,  even  though  manifest  in  minutest  traces, 
can  be  differentiated.  Similarly,  the  secret  of  blood  relationship  has  begun 
to  be  unravelled;  and  there  is  a  possibility  even  of  solving  the  problems  of 
metabolism. 

Closely  associated  with  serum  diagnosis  is  the  serum  therapy.  Even 
though  the  general  application  of  the  latter  is  not  as  widely  developed  as 
that  of  the  former,  it  must  be  remembered  that  through  this  medium  diph- 
theria has  been  transformed  from  a  fatal  to  a  combatible  disease,  and 
incidentally  made  the  name  of  Behring  immortal.  To-day,  attempts  are 
constantly  being  made  to  treat  other  bacterial  and  toxic  diseases  by  specific 
therapy  and  it  is  to  be  hoped  that  success  will  soon  be  met  with. 

The  study  of  serum  therapy  and  serum  diagnosis  is  undertaken  in 
various  ways.  It  is  comparatively  simple  to  learn  only  the  purely  technical 
details.  All  large  laboratories  have  trained  assistants  for  the  performance 
of  certain  reactions  or  groups  of  reactions  with  absolute  precision.  Although 
as  we  have  said,  they  do  such  work  as  assigned  to  them,  with  accuracy,  they 
are  nevertheless  far  from  a  thorough  understanding  of  the  subject  of  serum 
diagnosis.  Unfortunately  this  blind  method  of  procedure  has  recently 
been  advocated  to  an  alarming  extent.  In  addition,  the  practical  success 
which  the  Wassermann  reaction  has  met  with,  has  inculcated  the  desire  in 
certain  schools  of  physicians,  for  the  carrying  out  of  this  test  alone,  and  thus 
to  become  independent  of  the  use  of  large  laboratories.  To  meet  this 
demand,  short  courses  have  been  established  and  the  serum  diagnosis  of 
syphilis  taught  with  lightning  rapidity.  That  such  a  state  of  events  is 
absolutely  injurious  is  clearly  evident.  It  is  impossible  for  one  to  be  a 
specialist  in  a  certain  reaction  and  at  the  same  time  be  ignorant  of  the  other 


CONCEPTION    OF   IMMUNITY.  3 

phases  in  the  study  of  immunity.     Unreliable  and  erroneous  results  are  the 
inevitable  outcomes  of  such  unscientific  work. 

The  plan  followed  in  this  book  consists  in  taking  up  all  of  the  important 
principles  and  methods  of  immunity,  even  though  at  present  some  may 
attract  no  direct  practical  attention.  The  principle  of  the  now  widely 
important  Wassermann  reaction  had  been  described  years  previously  by 
Bordet  and  Gengou,  but  merely  from  a  purely  theoretical  standpoint. 
Only  with  the  development  of  the  Wassermann  test,  did  it  attain  its  ~prac- 
tical  importance. 

To  start  systematically,  it  is  necessary,  primarily,  to  under- 
Conception     stand   certain   terms   frequently   employed.     First,  the  word 
of  Immunity,    immunity,  requires  explanation: 

After  an  individual  has  recovered  from  an  infectious  disease, 
he  passes  into  a  state  where  he  is  less  or  even  not  at  all  susceptible  to  the 
same  infection,  although  no  macroscopical,  microscopical  or  chemical 
change  can  be  shown  to  have  taken  place  in  his  system.  This  condition  is 
one  of  immunity.  And  as  the  body  itself  by  its  own  struggle  with  the  invad- 
ing bacteria  has  brought  about  this  immunity,  it  is  known  as  "  active  immun- 
ity." Jenner  and  Pasteur  have  employed  this  mode  of  immunity  acquired 
spontaneously  with  the  overcoming  of  an  infection  in  then*  principle  of 
prophylactic  vaccination.  The  exact  nature  of  this  active  immunity  is  only 
partially  understood.  It  can  be  shown,  however,  that  the  individuals 
thus  actively  immunized  have  within  their  organism  reaction  bodies  of  a 
specific  nature  directed  against  the  infecting  elements  and  their  poisonous 
products.  These  reaction  bodies  which  circulate  mainly  in  the  blood  serum, 
are  known  as  Antibodies. 

The  antibodies  are  of  different  classes  depending  entirely  upon  their 
varied  forms  of  activity.  While  some,  such  as  the  agglutinins  and  preci- 
pitins  have  the  property  of  grouping  their  respective  invading  agents 
into  small  clumps  or  precipitates  without,  however,  at  the  same  time 
embracing  protective  powers,  there  are  other  antibodies  which  act,  essen- 
tially, for  the  defense  of  the  organism  whether  by  neutralizing  the  poison 
of  the  bacteria  (antitoxin)  or  by  destroying  the  bacteria  (bacteriolysin) ,  , 
or  so  altering  the  bacteria  that  the  latter  can  be  more  easily  destroyed 
by  the  cells  (bacteriotropin,  opsonin).  The  last  three  types  of  immunity 
can  be  designated  respectively,  as  antitoxic,  bactericidal,  and  cellular 
immunity.  Naturally  there  are  many  intermediate  forms.  It  is  very 
probable  that  besides  these  well  recognized  forms  of  immunity  there 
may  be  others,  still  unknown.  Cellular  immunity  must  surely  have  a  far 
greater  range  of  importance  than  is 'at  present  ascribed  to  it.  There  is,  no 
doubt,  a  distinct  cell  immunity  which  acts  without  the  aid  of  any  serum  sub- 
stance and  is  known  as  "Tissue  Immunity"  ("histogene"  Immunitat). 

If  the  serum  of  an  animal  which  has  been  immunized  and  containing 


4  INTRODUCTION. 

antibodies,  is  injected  into  another  normal  but  non-immunized  animal,  the 
latter  acquires  the  power  of  being  immune  against  the  specific  infective 
agent.  In  this  case  the  immunity  was  not  established  by  direct  cell  activity 
on  the  part  of  the  animal,  for  the  organism  remained  passive,  and  had,  as  it 
were,  immunity  thrust  upon  it.  This  form  of  immunity  in  contradistinction 
to  "active  immunity"  is  designated  as  " passive  immunity." 

The  forms  of  immunity  thus  far  mentioned  were  all  "acquired"  either 
by  the  spontaneous  recovery  from  the  infection  or  the  artifical  transmission 
of  the  curative  antibodies.  In  contrast,  however,  to  this  "acquired"  immu- 
nity there  is  a  "natural"  immunity  by  which  is  understood  that  some  ani- 
mal species  are  not  at  all  susceptible  to  certain  infections.  Thus  man  has  a 
natural  immunity  against  a  group  of  diseases  markedly  fatal  for  some  of  the 
lower  animals,  e.g.,  chicken-cholera  and  hog-cholera.  That  this  natural 
immunity  is  almost  always  cellular  in  character  is  undeniably  true;  and  the 
most  important  form  of  this  natural  armament  against  infection  is  the  pow- 
erful leucocyte,  capable  of  engulfing  and  destroying  the  invading  enemy.  In 
other  words,  phagocytosis. 

Finally  one  should  speak  of  a  "local"  and  "general"  immunity,  mean- 
ing to  express  thereby  the  different  resistance  and  susceptibility  that  various 
organs  of  the  same  individual  display;  and  also  of  a  "relative"  and  "abso- 
lute" immunity  in  order  to  differentiate  quantitatively  a  transitory  immu- 
nity from  one  that  is  of  long  duration. 

Another  term  very  often  employed  is  "antibody."     This,  as 
Conception    kas  a}reac[y  been  explained,  is  a  name  used  to  designate  the 
Antibod       sPecinc  bodies  which  the  organism  produces  as  a  reaction 
against  the  infecting  agents  and  their  toxic  products.     Anti- 
bodies are  also  formed  when  animals  are  injected  with  foreign  proteids  not 
of  bacterial  origin,  such  as  the  blood  of  a  different  species  of  animal,  egg 
albumin,  etc.     In  order  that  these  antibodies  may  be  obtained,  the  sub- 
stances employed  must  enter  the  system  "parenteral,"  i.e.,  some  way  out- 
side of  the  gastrointestinal  tract. 

In  older  literature  the  terms  antibody  and  protective  body  were  used 
synonymously.  That  is  decidedly  incorrect,  inasmuch  as  not  all  antibodies 
possess  the  power  of  protection  and  not  every  actively  immune  organism, 
demonstrable  antibodies.  Furthermore,  antibodies  as  the  bacteriolysins 
which  are  generally  considered  to  have  protective  powers,  and  correctly  so 
too,  can  exist  in  a  system  in  large  numbers  without  necessarily  rendering 
that  organism  immune. 

As  an  example  of  how  complicated  various  chapters  in  the  study  of  immunity  can  be, 
will  be  clearly  evidenced  by  a  few  of  the  author's  experiments  with  the  hog-cholera 
bacillus.  Rabbits  rendered  actively  immune  by  inoculation  with  extracts  of  hog-cholera 
bacilli  possess  a  serum  which  when  injected  into  an  animal  of  a  different  species  as  the 
guinea-pig,  will  render  the  latter  passively  immune.  If,  however,  the  serum  is  injected 


THE    LAWS    OF    SPECIFICITY.  5 

into  another  animal  of  the  same  class  (another  rabbit),  no  protective  power  is  transmitted. 
In  other  instances  it  was  shown  that  the  rabbit  which  was  being  treated  with  the  purpose 
of  active  immunity  was  in  reality  never  immune,  as  it  always  succumbed  when  injected 
with  living  bacteria  even  though  its  serum  contained  bodies  which  were  perfectly  able  to 
passively  protect  guinea-pigs  against  the  same  deadly  infection. 

Just  as  it  is  incorrect  to  consider  an  antibody  and  protective  body  as  one 
and  the  same  thing,  it  is  equally  erroneous  to  deny  the  existence  of  protective 
bodies,  because  their  presence  cannot  be  demonstrated  by  a  certain  method 
of  laboratory  examination.  It  must  be  kept  in  mind  that  there  are  still 
many  unsolved  problems  in  the  subject  of  immunity,  and  that  therefore 
only  the  positive  findings  should  be  the  basis  for  drawing  conclusions. 

In  order  to  learn  the  nature  of  these  antibodies  attempts  have  been  made 
to  isolate  them  chemically.  Thus  far  all  such  trials  have  been  unsuccessful. 
It  is  even  uncertain  whether  these  so-called  antibodies  are  definite  chemical 
entities.  Only  the  effects  of  the  serum  as  a  whole  are  known,  and  the 
ingredients  in  it  to  which  these  activities  are  attributed  are  thought  of  as 
antibodies.  For  didactic  purposes  antibodies,  as  antitoxins,  agglutinins, 
etc.,  will  be  spoken  of  in  this  book  when  the  antitoxic  or  agglutinating  prop- 
erties exclusively,  are  meant. 

In  spite  of  the  individual  differences  which  are  ascribed  to  the 
The  Law  of  various  classes  of  antibodies,  there  is  one  quality  possessed  by 
Specificity,  all — their  specificity.  To  explain  this  by  a  rather  crude 
example,  may  be  mentioned  the  fact  that  typhoid  antibodies 
will  give  their  various  reactions  of  immunity  only  when  these  are  performed 
with  the  typhoid  bacillus,  and  cholera  antibodies  only  when  performed  with 
the  cholera  vibrio.  Substances  which  lack  this  essential  property  of  speci- 
ficity cannot  be  considered  antibodies,  although  they  may  fulfill  all  other 
requirements.  There  are  indeed  limitations  to  this  fast  rule,  but  these  will 
be  considered  subsequently.  For  the  present  the  following  can  be  taken  as 
a  fixed  fact;  namely,  that  every  true  antibody  is  absolutely  specific,  and  that 
all  substances  or  bodies  which  are  not  specific  cannot  be  real  antibodies. 
The  law  of  specificity  is  the  fundamental  principle  of  serum  diagnosis.  As 
soon  as  the  specificity  of  a  reaction  becomes  doubtful,  its  diagnostic  im- 
portance suffers  greatly.  In  the  following  pages,  therefore,  the  question 
whether  or  not  a  reaction  is  specific  will  be  repeatedly  discussed,  and  it  will 
be  the  aim  in  every  way  possible,  especially  by  the  use  of  control  tests  and 
experiments  to  outline  the  limits  of  this  specificity.  Here,  even  at  so  early  a 
stage  of  the  discussion,  the  absolute  necessity  of  these  control  tests  must  be 
urged,  even  though  it  may  appear  superfluous  to  the  beginner,  when  for 
apparently  simple  experiments  controls  are  performed  which  consume  more 
time  than  the  actual  diagnostic  test  itself.  Probably  also  the  desire  will 
arise,  and  perhaps  be  satisfied,  to  omit  these  control  experiments.  This 
done,  notwithstanding  of  a  possibility  to  obtain  for  even  a  long  time,  per- 


6  INTRODUCTION. 

fectly  good  results,  it  cannot  be  too  often  or  too  emphatically  impressed  upon 
all  workers  in  immunity  methods,  that  the  only  guard  against  mistakes  and 
failures  in  diagnosis  is  necessarily  found  in  control  tests.  And  especially 
in  doing  research  work,  the  latter  are  indispensable.  For,  experimental 
work  which  involves  reasonable  possibilities,  or  has  any  pretension  toward 
plausibility  warrants  no  true  scientific  conclusion  without  the  employment 
of  such  tests. 

The  author  has  made  it  a  rule,  whenever  new  findings  in  serum  diagnosis  are  published, 
always  to  look  for  the  given  control  experiments.  If  these  are  insufficient,  then  no  matter 
what  the  contents  may  be,  the  value  of  the  research  is  slight,  for  all  its  claims  only  may, 
but  not  necessarilv  must,  be  correct. 


CHAPTER  II. 
LABORATORY  EQUIPMENT. — GENERAL  TECHNIQUE. 


Although  some  of  the  tests  of  serum  diagnosis  are  comparatively  simple 
and  can  be  performed  in  one's  office  or  even  at  the  bedside,  in  most  instances 
a  laboratory  equipment  is  essential.  This  of  course  does  not  at  all  imply 
the  necessity  of  such  elaborate  apparatus  as  one  is  accustomed  to  find  in  our 
present  up-to-date  bacteriological  or  serological  laboratories  where  a  great 
deal  of  complicated  research  work  is  done.  For  the  practical  application  of 


FIG.   i. — A  room  in  the  laboratory  of  the  Royal  Institute  of  Berlin  for  the  study  of 

infectious  diseases. 

serum  diagnosis,  as  employed  at  the  hospital  or  in  private  practice,  an  outfit 
much  less  costly  is  perfectly  sufficient.  As  regards  the  question  of  a  room, 
the  selection  of  one  with  two  windows,  allowing  the  entrance  of  sufficient 
light,  is  indispensable.  At  the  same  time,  however,  some  arrangement 
should  be  made  in  connection  with  the  windows  in  order  that  the  direct  rays 

7 


8  LABORATORY   EQUIPMENT. 

of  the  sun  be  prevented  from  striking  one's  desk.  Strong  sunlight  may 
weaken  or  even  destroy  the  virulence  of  cultures,  or  bring  about  many 
changes  in  sera.  Even  diffuse  daylight  should  not  be  considered  as  entirely 
inert.  A  general  rule  to  be  remembered  is  never  to  expose  any  biological 
reagent,  be  it  a  bacterial  culture  or  any  form  of  its  derivative,  a  serum,  or 
any  other  substance  to  daylight  any  longer  than  is  absolutely  necessary. 
If  this  dictum  is  followed,  one  will  avoid  many  a  difficulty. 

To  conform  with  this  idea,  it  is  wise  to  have  upon  the  table  a  small  closet  into  which 
the  cultures  and  sera  can  be  placed  for  the  time  that  they  are  being  used.  Such  a  con- 
venient receptacle  can  be  made  out  of  a  large  cigar  box,  painted  black  inside  and  out, 
with  its  lid  replaced  by  a  small  black  curtain. 

The  table  or  desk  at  which  one  works  should  be  near  the  window,  and  covered  with 
filter-paper,  upon  which  should  come  a  glass  or  asbestos  plate.  Instead  of  a  wooden 
table  it  is  certainly  more  elegant,  but  costlier  to  have  a  top  plate  of  glass.  Upon  the 
table  there  should  be  a  Bunsen  burner,  a  microscope,  a  lamp  for  microscopic  work  at 
night,  a  dish  filled  with  sublimate  or  cresol  into  which  the  infected  substances,  old 
cultures,  used  pipettes  and  graduates  are  placed. 

It  is  very  convenient  to  have  running  water  and  a  hood  in  the  same  room.  Still 
neither  of  these  is  absolutely  necessary.  As  for  larger  apparatus — must  be  mentioned, 

a  thermostat,  a  mechanism  for  shaking,  a  dry 
sterilizer,  a  good  autoclave,  a  water-bath,  an  in- 
strument sterilizer,  a  water  or  electrical  centrifuge, 
an  ice  chest,  a  closet  for  instruments  and  glass- 
ware, and  finally  animal  cages  of  the  kind  that  are 
easily  cleansed. 

As  for  instruments  and  glassware  the  following 
are  required:  scalpels,  scissors,  forceps,  glass- 
cutter,  sterilizable  syringes  of  various  sizes,  grad- 
uates of  10,  25,  100,  and  loooc.  cm.  capacity  each, 
pipettes  of  i  c.  cm.  with  i/ioo  divisions  and  pipettes 
of  10  c.  cm.  with  i/io  divisions,  a  sterilizable 
FIG.  2.— Instrument  (After  Czap-  pipette  retainer,  Erlenmeyer  flasks,  Petri  and 
Standardization  °f  Kolle's  dishes,  test-tubes,  dark  glass  flasks,  ordinary 
water  glasses,  funnels,  glass  tubes  of  various  sizes, 
and  test-tube  racks.  Furthermore,  a  platinum  needle  and  a  platinum  loop  are  required. 
For  making  a  loop  of  a  definite  size,  and  one  which  can  always  be  referred  to,  the  small 
instrument  devised  by  Czaplewski  is  of  great  help.  It  consists  of  four  round  metal  bars 
of  i,  2,  3,  and  5  mm.  in  diameter  around  which  the  platinum  wire  can  be  twisted  in 
order  to  make  a  standard  loop  (Fig.  2). 

All  instruments  and  glassware  used  for  serum  work  should  be  perfectly 
clean.  It  is  best  to  have  all  the  glassware  plugged  with  non-absorbent 
cotton,  and  sterilized  by  dry  heat.  It  is  never  advisable  to  clean  the  glass- 
ware with  strong  acids,  alkalies  or  other  strong  chemicals.  If  this  has  been 
done,  the  chemicals  must  be  thoroughly  removed  by  washing,  as  the  slightest 
trace  may  interfere  with  the  accuracy  of  some  tests. 

All  used  glassware  should  at  once  be  placed  into  a  disinfecting  solution. 


TECHNIQUE    OF   INOCULATION.  9 

For  this  purpose,  lysol,  lysoform  and  cresol  solutions  are  highly  recommend- 
able.  Sublimate  is  less  efficient  because  it  coagulates  albumins  and  thus 
may  lead  to  plugging  of  pipettes  which  may  have  contained  blood  rests.  If 
highly  infectious  material  has  been  examined,  it  is  best  to  place  the  entire 
disinfectant  solution  containing  the  used  glassware  into  the  autoclave, 
sterilize  it  there,  then  wash  the  supply  thoroughly  with  soap,  dry  and  resteril- 
ize  it  by  dry  heat  for  i  to  2  hours  at  120°  C. 

The  Technique  of  Inoculation. 

Both  for  serum  diagnosis  and  serum  therapy,  the  serum  is  required  from 
animals  which  have  been  artificially  immunized  against  the  bacteria  or 
their  products  of  secretion.  Almost  without  exception,  this  immunization 
is  produced  by  injecting  the  animal  with  the  infectious  virus.  The  method 
of  inoculation  is  either  intravenous,  w/raperitoneal,  or  subcutaneous. 

The  technique  of  intravenous  injection  varies  somewhat  with 
Intravenous  different  animals.  In  rabbits,  the  veins  running  along  the 
Injection.  outer  margins  of  the  ears  are  most  suitable.  The  assistant 
sits  upon  a  chair,  holds  the  hind  legs  and  body  of  the  rabbit 
tightly  fixed  between  his  knees  and  thus  has  his  hands  free  to  steady  the 
rabbit's  ears.  Another  method  consists  in  placing  the  rabbit  upon  the  table 


FIG.  3. — Intravenous  inoculation.     (After  Uhlenhuth.} 

and  firmly  holding  him  there  while  the  injection  is  made  (Fig.  3).  The  ear 
is  first  struck  gently  with  the  fingers  and  washed  with  alcohol  and  xylol. 
If  the  hair  is  very  long,  it  should  be  clipped.  If  the  vein  running  along  the 
outer  margin  of  the  ear  is  exceptionally  small,  it  can  be  made  more  promi- 
nent by  compressing  it  between  the  thumb  and  index  finger  at  the  root  of  the 


10  LABORATORY    EQUIPMENT. 

ear.  No  force  should  be  used  with  the  injections;  the  fluids  should  be 
allowed  to  flow  into  the  blood  stream  very  slowly.  Glass  syringes,  or  such 
as  can  be  sterilized  easily,  are  preferable.  Air  bubbles  are  to  be  carefully 
guarded  against  in  order  to  exclude  the  danger  of  air  embolism. 

If  infectious  material  is  used  for  injection,  it  is  advisable  in  such  in- 
stances, to  place  a  small  piece  of  cotton  moistened  in  alcohol  or  carbolic 
around  the  point  of  union  between  the  needle  and  the  barrel  of  the  syringe 
to  prevent  the  possible  escape  of  any  fluid  which  usually  occurs  at  this 
point. 

After  inoculation  is  completed,  the  needle  should  be  quickly  withdrawn, 
a  small  piece  of  non-absorbent  cotton  placed  upon  the  needle  puncture  and 
compression  applied.  If  non-virulent  bacteria  or  albumin  is  injected,  the 
bleeding  may  be  almost  instantly  controlled  by  firmly  squeezing  the  vessel 
above  the  puncture  wound  with  the  edge  of  one's  finger  nail. 

In  guinea-pigs  intravenous  inoculation  is  more  difficult,  as  here  there  are 

no  large  superficial  veins.     The  Jugular  or  Iliac  vein  is  therefore  chosen, 

and  must  be  dissected  free.     It  is  not  necessary  to  tie  off  the  vessel,  but  the 

wound  should  be  firmly  compressed  by  means  of  clean  gauze  or  cotton. 

Morgenroth   has   substituted   the   simpler   method   of  intra- 

Intracardial    car  dial  inoculation.     The  point  of  maximum,  pulsation  of  the 

Injection,     heart  to  the  left  of  the  sternum  is  made  out  by  palpation 

and  a  thin  sharp  needle  is  inserted  at  the  specified  area.     The 

spurting  of  blood  indicates  that  the  needle  is  within  the  heart.     Thereupon 

the  already  filled  syringe  is  carefully  fitted  on  to  the  needle  and  the  contents 

slowly  injected.     The  syringe  is  then  detached  from  the  needle  and  blood  is 

again  allowed  to  spurt  out  in  order  to  be  absolutely  convinced  that  the  needle 

is  still  in  the  heart.     It  is  next  quickly  withdrawn.     By  this  method  it  is 

possible  to  inject  about  11/2  c.c.  directly  into  the  blood  stream. 

In  dogs,  sheep,  goats,  horses,  etc.,  the  intravenous  injection  is  given  into  the  jugular 
vein  directly  through  the  skin  which  must  be  thoroughly  shaved,  cleaned  and  disinfected. 
Compression  by  the  finger  makes  the  vein  more  prominent. 

In  dogs  the  popliteal  vein  is  frequently  selected.  In  man  the  intravenous  injection  is 
given  into  one  of  the  veins  on  the  anterior  surface  of  the  elbow  joint. 

Several  general  rules  are  to  be  observed  when  giving  intravenous  inocu- 
lations. First  of  all,  only  small  quantities  of  fluids  should  be  injected; 
secondly,  the  temperature  of  the  fluids  for  injection  should  not  differ  from 
that  of  the  body;  thirdly,  substances  that  are  strongly  hemolytic  may  pro- 
duce marked  disturbances  or  even  sudden  death  of  the  animal;  fourthly, 
if  an  animal  is  to  be  frequently  inoculated  it  is  best  to  puncture  the  vein  for 
the  first  inoculations  as  far  peripherally  as  possible  and  give  each  subse- 
quent injection  more  centrally,  for  very  often  thrombi  are  formed  at  the  site 
of  inoculation. 


INTRAPERITONEAL    INJECTION. 


II 


INTRAPERITONEAL    injection    is    employed    most    frequently 
Intraperito-    among  rabbits  and  guinea-pigs.     The  main  danger  associated 
neal         with  this  method  is  possible  injury  to  the  intestines;  but  by 
Injection,     heeding  the  following  advice,  this  can  be  prevented.     The  ani- 
mal should  be  fixed  or  held  head  down.     In  this  position,  the 

loops  of  intestines  tend   to  sink  toward  the   diaphragm.     This  is  further 

helped  along  by  gentle  downward  massage  over  the 'abdomen  thus  leaving 

an  area,  above  the  bladder,  which  is  sometimes  free  from  intestines.-  Another 

protective     measure,     consists    in 

using  a  blunt  canula  which  can  be 

made   by   breaking  off   the   sharp 

point  of  the  needle.      As  it  is  at 

times  difficult  to  pierce   the   skin 

with  this  blunt   instrument,    it    is 

advisable    to    previously    make    a 

minute  incision  through  the  cutis 

and    subcutis    with   a   sharp   pair 

of   scissors   and    pass    the    needle 

through  this  small  opening.     The 

needle    should    not     be     plunged 

directly  into  the  peritoneal  cavity, 

because    at    the    withdrawal,    the 

injected  fluid  easily  escapes  through 

the  punctured  opening.     First,  it  is 

inserted    subcutaneously    upward, 

in  the  long  direction  of  the  animal ; 

then  the  hand  is  raised  and   the 

needle  forced  horizontally  forward 

through  the  peritoneum,  thus  leav- 
ing the  opening  in  the  peritoneum 

at  a  different  level  than  the  one 

through  the  muscles  and  fascia,  thereby  making  the  escape  of  fluid  more 

difficult.     One  readily  realizes  that  he  has  gone  through  the  peritoneum  by 

a  relaxation  of  the  reflex  abdominal  rigidity  (Fig.  4) . 

For  the  intraperitoneal  injection  in  guinea-pigs,  Friedberger  has  devised 

a    procedure    which    is    very   satisfactory   and   furthermore   does   away 

with  the  necessity  of  an  assistant.     It  can  also  be  employed  in  Pfeiffer's 

test  for  the  removal  of  exudates  from  the  peritoneal  cavity.     The  giunea- 

pig  is  allowed  to  creep  into  the  breast  pocket  of  the  laboratory  gown 

until  its  head  and  thorax  are  inside  of  the  pocket.      Its  hind  legs  are 

grasped   between   the   middle    and   ring   fingers    of    the    left   hand   and 

flexed  on  the  back,  thus  giving  a  free  exposure  of  the  lower  parts  of  the 

abdomen  (Fig.  5). 


FiG.  4. — Intraperitoneal  injection  of  rabbit. 
(After  Uhlenhuth.} 


12 


LABORATORY   EQUIPMENT. 


SUBCUTANEOUS  inoculation  is  the  simplest  of  all  methods. 

Subcutaneous  A  fold  of  skin  is  elevated  between  the  thumb  and  index  finger 

Injection,     of  the  left  hand  and  the  needle  plunged  into  the  subcutaneous 

tissue.     In  rabbits  and  guinea-pigs  the  skin  of  the  back  or 

abdomen  is  chosen,  as  the  subcutaneous  tissue  here  is  not  tense.     In  goats, 

sheep,  and  horses  the  skin  of  the  neck  and  shoulder  region  is  preferred. 


I  $i>- 


FIG.  5.  —  Removal  of  peritoneal  exudate.     Guinea-pig  held  in  Friedberger's  position.     (Origi 


The  skin  of  the  back  and  abdomen  is  to  be  avoided  because  following  the 
injection  edema  frequently  arises,  which  may  extend  to  the  lower  extremities 
and  thus  interfere  with  locomotion. 

If  abscesses  arise  after  subcutaneous  injection,  they  should  be  opened, 
washed  out  with  lysol  solution  and  dressed  with  iodoform. 

The  Methods  of  Obtaining  and  Preserving  Serum. 

Venesection  or  venous  puncture  is  the  method  best  adapted  for  obtaining 
blood  from  animals.     The  veins  employed  for  that  purpose  are  those  which 


WET    CUPPING. 


have  already  been  mentioned  in  connection  with  intravenous  injections. 
A  simple,  large,  hollow  needle  is  all  that  is  required.  Suction  with  a  syringe 
is  superfluous.  Only  in  Morgenroth's  method  of  removing  blood  directly 
from  the  heart  of  guinea-pigs  is  aspiration  necessary.  In  rabbits  enough 
blood  can  be  collected  by  making  an  incision  into  the  vein  along  its  long  axis, 
with  a  sharp  knife,  or  by  dividing  the  vein  transversely  with  the  scissors. 
The  blood  thus  collected  is  not  absolutely  sterile. 

In  man,  if  only  a  small  quantity  of  blood  is  required,  it  can  be  obtained 
from  the  finger  or  ear.  If,  however,  a  larger  amount  is  necessary,  puncture 
of  one  of  the  veins  in  the  bend 
of  the  elbow  with  the  Strauss 
canula  is  resorted  to.  It  goes 
without  saying  that  this  area 
must  be  properly  disinfected 
with  soap  and  water,  ether, 
alcohol  or  sublimate.  Wright's 
method  for  collecting  moderate 
quantities  of  blood  will  be  re- 
viewed in  the  chapter  on  opsonic 
studies. 

If  the  vein  is  prominent,  the 
canula  is  thrust  into  the  vein 
directly  through  the  skin.  Here 
the  author  has  found  it  more 
convenient  to  point  the  canula 
upward,  i.e.,  in  the  direction  of 
the  blood  stream.1  In  cases 
where  the  vein  does  not  stand 
out  it  can  be  made  to  do  so 
either  by  applying  pressure  with 
the  finger  upon  its  central  part  or  placing  a  tight  rubber  bandage  or  rubber 
tube  about  the  arm.  This  should  not,  however,  be  tight  enough  to  obliterate 
the  radial  pulse.  In  very  fat  individuals,  even  these  means  do  not  suffice 
so  that  the  vein  must  be  dissected  free  and  incised.  After  completion  of  the 
venesection  the  arm  is  elevated,  slight  pressure  made  upon  the  wound  with 
sterile  cotton  and  a  bandage  applied.  If  a  small  amount  of  blood  is  sufficient, 
and,  as  in  most  serological  examinations  absolute  sterility 

is  not  essential,  venesection  can  be  replaced  by  the  method 
Wet-cupping.      p 

of  wet-cupping.     For  this  procedure  a  scarifier  and  Bier  cup 

are  required.     The  technique  is  as  follows  (Fig.  7). 


FIG.  6. — Puncture  of  vein.     (Original.} 


1  The  editor  has  found  that  more  blood  is  obtained  by  thrusting  the  canula  into  the  vein  in 
the  reverse  direction. 


14  LABORATORY    EQUIPMENT. 

Some  part  of  the  skin  of  the  back  is  thoroughly  disinfected  and  a  well-fitting  Bier  cup 
firmly  applied.  Suction  arises  and  the  skin  assumes  a  dark  bluish-red  appearance. 
After  half  a  minute  the  cup  is  removed,  the  scarifier  applied  and  the  cutting  edges  set 
free.  The  scarifier  is  then  reapplied,  but  this  time  at  right  angles  to  the  previous  incisions 
and  the  edges  again  set  free.  Suction  is  again  made  by  the  Bier  cup  and  the  blood  is 
thus  forced  out  from  the  multiple  incisions. 


FIG.  7. — Obtaining  blood  by  the  wet  cupping  method. 

The  blood  obtained  by  any  of  the  above  methods  is  collected  into  a  sterile 
vessel  (graduate,  flask,  test-tube)  and  allowed  to  coagulate.  The  clot  is 
then  separated  from  the  sides  of  the  vessel  by  a  sterile  glass  rod  or  platinum 
needle,  the  vessel  plugged  with  absorbent  cotton  and  placed  into  the  ice- 
chest.  After  12  to  24  hours  the  serum  begins  to  separate  out  from  the  clot. 
If  the  serum  is  required  immediately,  the  blood  is  allowed  to  flow  directly 
into  centrifuge  tubes,  the  clot  separated  from  the  sides  and  the  tubes  centri- 
fugalized.  With  a  well  regulated  centrifuge  serum  appears  after  several 
minutes. 

There  are  several  rules  to  be  kept  in  mind  when  using  a  centrifuge. 
Rules  for      i.  The  machine  must  be  well  oiled. 

the  Use  of     2.  The  counterweights  must  be  absolutely  of  the  same  weight. 
Centrifuge.    3.  The  centrifuge  should  never  be  suddenly  stopped,  but  allowed  to  do 

so  of  its  own  accord. 

4.  In  starting  it,  the  motor  should  be  gradually  turned  on. 

5.  If  the  centrifuge  is  slightly  out  of  order  it  should  not  be  used,  but  repaired  at  once, 
otherwise  it  may  be  ruined  forever. 

6.  One  should  never  centrifugalize  with  cotton  plugs  in  the  test-tubes.     If  the  latter 
must  be  sealed,  rubber  stoppers  should  be  used. 


THERMOSTABILE    OR    THERMOLABILE    SUBSTANCES.  15 

The  color  of  a  serum  is  greatly  variable,  depending  mainly  upon  its 
hemoglobin  or  fat  content.  Blood  taken  at  the  height  of  the  period  of  diges- 
tion shows  a  chylous  serum.  The  serum  of  nursing  women  contains  milk, 
that  of  icteric  people  contains  bile.  For  most  serological  examinations 
these  elements  in  the  serum  are  inert,  and  do  not  interfere  with  the  reading 
of  the  results.  In  precipitin  reactions,  however,  the  serum  must  be  abso- 
lutely clear. 

If  serum  is  to  be  kept  for  a  long  time,  there  are  several  ways  that  it  may 
be  retained  without  losing  its  activity.  The  method  chosen  depends  upon 
the  serum  substance,  which  is  important. 

As  will  be  pointed  out  again,  substances  are  either  thermostabile  or 
thermolabile.  The  preservation  of  thermostabile  substances  (agglutinins, 
amboceptors)  is  usually  very  simple.  It  is  sufficient  to  place  the 
clear  serum  which  has  separated  from  the  clot,  into  a  sterile 
test-tube  plugged  with  absorbent  cotton,  and  to  put  it  into  the 
ice  chest  away  from  the  light.  To  reassure  its  perfect  preserva- 
tion one  may  add  to  it  some  phenol  in  such  proportion  that  the 
carbolic  should  be  present  to  the  extent  of  1/2  per  cent,  solution, 
e.g.,  to  nine  c.c.  of  serum  add  i  c.c.  of  a  5  per  cent,  phenol  solu- 
tion. The  latter  should  be  added  drop  by  drop  and  agitated, 
so  as  to  avoid  the  formation  of  precipitates.  Another  method, 
which  the  author  employs  almost  exclusively  for  the  preserva- 
tion of  amboceptor  containing  sera,  consists  simply  in  inactivating 
the  sera  at  56°  C.  for  a  half  hour  and  then  placing  them  into  the 
ice  chest.  Inactivation  has  the  advantage  of  stopping  molecular 
changes  which  are  produced  by  ferment  actions  of  fresh  serum. 
Furthermore,  heating  acts  as  a  sterilizer  for  isolated  air  germs 
which  may  have  found  their  way  into  the  serum  during  the 
process  of  getting  it.  In  this  form,  a  serum  can  be  kept  in  the 
ice  box  for  several  weeks  without  any  material  change.  Occa-  FIG  g. 
sionally  one  finds  that  a  serum  will  undergo  contamination  in  rTllbe  used 

J  °  f  for  preserva- 

spite  of  inactivation,  so  that  it  follows,  that  if  a  serum  is  to  be  pre-      tion  of 


served  for  several  months,  it  is  advisable  to  seal  it  in  a  test  tube, 
For  this  purpose  a  brown  glass  tube  slightly  drawn  out  at  its 
upper  end  is  employed  (Fig.  8).  The  serum  is  placed  into  this  sterilized 
tube  and  then  the  latter  is  sealed  in  the  flame  at  its  narrow  part.  Bacterial 
and  organ  extracts  are  well  kept  in  this  way.  The  best  method  of 
preservation  consists  in  evaporating  the  serum  to  dryness  in  a  vacuum 
desicca  or.  This  procedure  is  rather  complicated  and  can  therefore  be 
employed  only  in  institutions. 

A  vacuum  desiccator  with  beatable  plates  is  used.  The  serum  is  poured  in  very  thin 
layers  into  sterile  flat  dishes  and  allowed  to  dry  out  in  the  desiccator  at  a  temperature  of 
30°  C.,  later  on  at  35°  C.  in  a  vacuum  of  3  cm.  mercury.  The  dried  serum  forms  a 


i6 


LABORATORY   EQUIPMENT. 


yellowish-red  horny  mass  which  is  scraped  off  from  the  dish  and  ground  up  in  a  mortar 
into  a  yellowish  powder.  The  serum  powder  is  then  placed  into  a  brown  glass  tube  and 
sealed. 

When  this  dried  serum  is  to  be  used,  the  tip  of  the  ampulla  is  broken  off,  and  several 
drops  of  isotonic  salt  solution  at  a  temperature  of  30°  are  poured  in,  in  just  sufficient 
an  amount  to  moisten  the  wall  of  the  glass  tube.  By  rolling  the  tube  to  and  fro,  one  finds 
that  the  serum  powder  will  easily  stick  to  the  moistened  wall.  The  granules  are  allowed 
to  swell  up  and  after  they  have  done  so,  enough  isotonic  salt  solution  is  added  to  make  up 
the  original  volume. 

For  the  preservation  of  thermolabile  substances,  the  method  of  freezing  has  been 
suggested.  Morgenroth  has  devised  for  this  purpose  a  simple  and  handy  apparatus 
named  Frigo  which  can  be  obtained  fromLautenschlager,  Berlin.  Although  for  most  tests 
this  method  of  preservation  has  been  employed  with  success,  Neisser's  clinic  reports  that 
sera  preserved  in  the  Frigo  with  the  idea  of  retaining  their  complement  did  not  give  as 
accurate  complement  fixation  experiments  as  did  similar  fresh  sera. 

Friedberger  advises  the  addition  of  8  per  cent,  salt  solution  for  the  preservation  of  the 
complement.  When  the  serum  is  to  be  used  it  is  diluted  tenfold  with  distilled  water,  so 
that  a  10  per  cent,  dilution  of  complement  is  obtained.  By  the  addition  of  the  salt,  the 
resistance  against  the  harmful  effects  of  light,  room,  body  temperature,  and  chemical 
substances  like  phenol  is  increased,  but  the  thermolability  of  the  complement  remains  the 
same.  Drying  of  a  serum  in  a  desiccator  is  not  to  be  advocated  for  the  preservation  of 
the  complement  as  during  such  procedure  a  portion  of  the  complement  is  lost.  Once  the 
serum  is  in  its  dried  form,  however,  the  remaining  complement  is  retained  and  in  addition, 
has  become  resistant  against  high  heat. 

Filtration  of  Bacteria. 

It  is  important  in  many  serological  studies  to  be  able  to  separate  bacteria 
from  their  fluid  media  or  suspension.  This  is  accomplished  either  by 
centrifugalization  or  filtration.  The  first  method  does  not  completely  free 


FIG.  9. 

Pukal-filter. 


FIG.  10. — Filtration  through  a 
Pukal-filter. 


the  fluid  of  its  bacteria,  but  if  this  is  desired  the  method  of  filtration  is  essen- 
tial. In  this  connection,  however,  one  must  bear  in  mind,  that  many  albu- 
menous,  or  albumen-like  substances,  few  colloids  and  even  some  toxins,  do 
not  pass  the  filters  and  are  therefore  held  back.  Bacterial  filtration  is 
simplified  by  preliminary  centrifugalization  or  passing  the  fluid  through 


filter-pai 


FILTRATION    OF    BACTERIA. 


Iter-paper.  Different  porous  materials  have  been  used  for  bacterial  niters; 
of  which  especially  suitable  are  porcelain,  infusorial  earth  and  asbestos.  The 
nitration  apparatus  consists  of  the  respective  filter  and  the  receptacle  which 
receives  the  filtrate.  Filtration  takes  place  by  differences  in  pressure,  where 
either  the  fluid  is  forced  through  by  high  pressure  or  sucked  through  by  a 
vacuum  formed  in  the  receiving  vessel.  The  following  are  some  of  the 
filters  most  commonly  in  use. 

1.  CHAMBERLAIN'S  CYLINDER  FILTER,  F,  used  in  the  Pasteur  Institute  at  Paris.     The 
filter  cylinder  is  made  of  infusorial  earth  and  may  be  attached  to  any  faucet. 

2.  PUKAL  FILTER,  made  of  burnt  kaolin,  is  used  especially  for  the  nitration  of  large 
quantities  of  fluid.     The  filter  b  is  placed  into  the  beaker  e  containing  the  toxin  and 
bacterial  fluid.     The  former  is  then  closed  by  a  rubber  stopper,  perforated  by  a  central 
opening  through  which  runs  a  glass  tube  bent  at  right  angles,  and  this  in  turn  is  connected 
with  either  an  air  or  water  pump  for  producing  a  vacuum  inside  of  the  filter.     Between 
the  filter  and  vacuum  pump  can  be  interposed  a  sterile  jar  a.  (Figs.  9  and  10). 


FIG.  ii.— Relchel  filter. 


FIG.  12. — Lilliputian  filter. 


3.  THE  REICHEL  FILTER  (Fig.  n)  consists  of  a  glass  receiver  A,  having  a  side  neck  c 
and  at  the  bottom  a  tube-like  outlet  d.     A  porcelain  filter  B  fits  into  the  glass  jar  and 
rests  upon  the  margin  of  the  flask  by  means  of  a  broad  collar.     The  point  of  junction  is 
made  air  tight  by  means  of  a  rubber  cap  with  a  central  opening,  through  which  the 
cylinder  can  be  filled.     When  in  use  d  is  shut  off  by  a  rubber  tube  with  a  pinch  cock  and 
c  in  which  lodges  a  small  piece  of  cotton  is  connected  with  a  water  pump  that  is  instru- 
mental in  bringing  about  a  vacuum.     The  function  of  d  is  to  allow  the  removal  of  samples 
of  the  filtrate  and  finally  to  obtain  the  entire  filtrate. 

4.  THE  LILLIPUTIAN  FILTER,  candle-like  in  shape,  and  made  of  infusorial  earth,  is 
employed  for  the  filtration  of  very  small  quantities.     The  filter  is  cemented  upon  a  metal 
tube  which  is  screwed,  so  that  it  is  air  tight,  into  a  well-fitting  glass  cylinder  open  at  the 
top.     The  tube  is  passed  through  a  rubber  cork  which  tightly  closes  an  exhaust  flask. 


i8 


LABORATORY    EQUIPMENT. 


The  fluid  to  be  filtered  is  placed  into  the  glass  cylinder  and  sucked  through  into  the  flask 
by  means  of  a  vacuum  which  is  here  produced.  For  the  purpose  of  collecting  very  small 
quantities  a  test-tube  may  be  placed  into  the  exhaust  flask  (Fig.  12). 

Preparation  of  Dilutions  and  Measurement  of  Small  Amounts   of 

Bacteria. 

All  serological  methods  are  to  be  considered  on  quantitative  bases.  In 
serum  diagnosis  as  well  as  in  the  therapy,  the  amount  of  the  serum  employed 
is  the  deciding  factor.  Similarly,  the  number  or  amount  of  bacteria  required 
either  for  the  purposes  of  immunization  or  serological  reactions  is  of  extreme 
importance. 

One  cubic  centimeter  is  the  unit  of  measure  for  serum  and  all  fluid  material 
(Bouillon  cultures,  exudates,  etc.).  //  small  quantities  are  required,  it  is 
best  to  dilute  the  fluid  with  0.85  per  cent,  saline  solution.  The  exact  preparation 
of  dilutions  is  one  of  the  most  essential  technical  procedures  of  all  serum  diagnosis. 
Some  general  rules  may  be  of  help. 

Never  should  amounts  less  then  o.i  c.c.  be  measured  out  directly.  For 
beginners  even  o.i  is  best  measured  in  the  form  of  a  dilution,  as  errors  are 
apt  to  occur  very  easily. 

2.  The  decimal  system  should  be  adhered  to  as  much  as  possible. 

3.  The  dilution  should  be  made  just  before  it  is  to  be  used,  inasmuch  as 
many  substances  retain  their  activity  best,  or  only,  in  concentrated  form. 

The  following  is  an  example  of  correct  forms  of  dilution: 


Toxin. 

Dilution  of  toxin, 
i  :  10 

Dilution  of  toxin, 
i  :  100 

Dilution  of  toxin, 
i  :  1000 

0.  I  C.C.   = 

I         C.C. 

0.05  c.c.  = 

O.OI  C.C.   = 

o.  5  c.c. 

O.  I   C.C. 

=  I         C.C. 

0.005  c-c-  = 

O.OOI  C.C.   = 

0.5  c.c. 

0.  I   C.C. 

=  1.0  C.C. 

0.0005  c'c-  = 

0.5  c.c. 

The  stock  dilution  of  i  :  10  is  made  by  measuring  off  i  c.c.  of  toxin  an  adding  9  c.c. 
of  0.85  per  cent,  of  saline. 

The  dilution  i  :  100  can  be  made  by  taking  i  c.c.  of  toxin  and  adding  99  c.c.  of  saline. 
It  is  more  practicable,  however,  to  take  i  c.c.  of  the  i  :  10  stock  dilution  and  add  9  c.c. 
of  saline.  If  the  dilution  i  :  10  is  not  present  and  only  a  small  amount  of  the  dilution 
i  :  100  is  desired,  the  latter  is  made  by  taking  o.i  toxin  :  10.0  NaCl  sol.  Similarly 
i  :  1000=0.1  :  100=  i  c.cm.  of  the  dilution  (i  :  10)  :  100=  i  c.c.  of  the  dilution  (i  :  100)  : 
10.0. 


PREPARATION    OF    DILUTIONS. 
The  following  table  shows  the  details  of  various  dilutions: 


Dilution  i  :  10. 


Dilution  i  :  100. 


Dilution  i  :  1000.          Dilution  i  :  10,000. 


i  c.c.  +  9  c.c.  NaCl  sol.    i  c.c.  +  99  c.c.  NaCl  sol. 

i  c.c.  +  999  NaCl 

o.  1  +  999.9  NaCl 

=  o.  c.c.  +  0.9  c.c.  NaCl 

=  o.  ic.  c.  +  9.9  c.c.  NaCl 

=  o.  1  +  99.9  NaCl 

=  i  c.c.  of  dilution 

solution. 

solution. 

i  :  1000 

=  0.2  c.c.  +  1.8  c.c.  NaCl 

=  0.2  +  19.  8  c.c.  NaCl 

0.2  c.c.  +  199.  8            +  9  c.cr-of-NaCl 

solution. 

solution. 

=  2  c.c.  +  1998  c.c. 

=0.3  c.c.  +  2.7  c.c.  NaCl 

=  0.3  +  29.7  c.c. 

=  i  c.c.  of  dilution        =0.1  c.c.  of  dilution 

solution. 

i  :  100  +  9  c.c.  NaCl 

i  :  100 

=  o.  i  c.c.  dil.  i  :   10 

+  9.  9  c.c.  NaCl 

=  3  c.cm.  +  27  c.c.           =i  c.c.  of  dil.  i  :  10 

+  9.9  c.c.  NaCl 

=  i  c.c.  dil.  i  :  100 

NaCl                      +  9  c.c.  of  NaCl.  sol. 

=  0.1  c.c.  of  dil. 

+  99  c.c.  NaCl. 

i  :  100  +0.9  c.c. 

=  o.i  c.c.  dil.  i  :  10. 

=  10  c.cm.  +  90  c.c.           =  2  c.c.  of  dil.  i  :  10 

NaCl  solution. 

+  99.9  c.c.  NaCl. 

NaCl. 

+  18  c.c.  NaCl.  sol.,  etc. 

In  carrying  out  these  dilutions,  it  is  best  to  measure  off  the  small  quantities  o.i—i  c.c. 
with  a  pipette;  allow  this  to  run  into  a  well-graduated  measuring  glass  and  add  enough 
saline  to  make  the  required  dilutions.  For  example,  if  30  c.c.  of  a  dilution  i  :  100  is 
desired,  .3  c.c.  should  be  measured  off  with  a  pipette  and  allowed  to  flow  into  a  50  or  100 
c.c.  graduated  cylinder  and  saline  solution  added  up  to  30.0  c.c. 

It  should  always  be  one's  aim  to  get  along  with  small  quantities  of  the  substance  to  be 
diluted.  If,  for  example,  8  to  10  c.c.  of  a  toxin  dilution  i  :  100  are  required  o.i  c.c.  of 
toxin+9.9  c.c.  of  saline  should  be  taken  and  not  i  c.c.  of  toxin  and  99  c.c.  of  NaCl  sol. 

Before  making  any  dilution  one  should  always  calculate  the  total  amount  of  substance 
required;  as  for  example  in  the  following  experiment: 


1.  Animal  o.  i    c.c. 

2.  Animal  0.05  c.c. 

3.  Animal  o.oi  c.c. 

4.  Animal  o.ooi  c.c. 


Toxin  subcutaneously 
Toxin  subcutaneously 
Toxin  subcutaneously 
Toxin  subcutaneously 


Here,  the  total  quantity  of  toxin  necessary,  is  found  by  adding,  to  be  0.161  c.c.  This 
represents  the  minimum  amount.  It  is  always  advisable  to  make  an  allowance  for  some 
loss  and  at  the  same  time  bring  up  the  amount  to  a  round  or  even  number.  0.2  c.c.  of 
toxin  would  fulfill  all  these  requirements.  This  amount  is  measured  off  by  a  pipette, 
placed  into  a  graduated  cylinder  and  saline  added  up  to  2.0  c.c.,  making  a  dilution  of 
i  :  10.  Then  0.2  of  this  dilution  (i  :  10)  is  taken,  placed  into  another  graduate,  and 
again  diluted  with  saline  up  to  2.0  thus  making  a  dilution  of  i  :  100.  The  above  problem 
therefore,  of  injecting  the  various  animals,  can  be  completed  as  follows: 

i.  Animal  receives  i      c.c.  of  dilution  i  :  10 
i.  Animal  receives  0.5  c.c.  of  dilution  i  :  10 

3.  Animal  receives  i      c.c.  of  dilution  i  :  100 

4.  Animal  receives  o.  i  c.c.  of  dilution  i  :  100 

The  unit  for  measuring  the  amount  of  bacteria  grown  upon  a  solid  me- 
dium is  represented  by  a  standard  sized  loop.  This  platinum  loop  takes  up 


20  LABORATORY    EQUIPMENT. 

about  2  mg.  of  bacterial  substance.     It  is  prepared  as  explained  in  Fig.  2. 
If  smaller  amounts  of  bacteria  are  used,  dilutions  must  be  made. 

For  instance,  1/4  of  a  loopful  of  bacteria  is  desired;  i  loopful  is  suspended  in  i  c.c. 
of  saline  and  3  c.c.  of  saline  added.  As  a  result,  i  c.c.  of  this  emulsion  contains  1/4 
of  a  loopful  of  bacteria.  If  1/16  of  a  loopful  is  necessary  i  c.c.  of  the  above  dilution  is 
added  to  3  c.c.  of  saline,  thus  making  i  c.c.  of  this  mixture  contain  1/16  of  a  loopful  of 
bacteria. 


CHAPTER  III. 
ACTIVE  IMMUNIZATION. 

IMMUNIZATION  WITH  LIVING  AND  DEAD  VIRUS. 

Active  immunization  depends  upon  the  principle,  that  an  organism  in 

overcoming  a  slight  infection  either  naturally  or  artificially 

The  Principle  acquired,  develops  enough  protective  bodies  to  withstand  a 

of  Active     similar,  severer,  natural,  or  acquired  infection.     Moreover, 

Immunization,  ft  serves  primarily  the  purpose  of  prophylaxis.     In  laboratories, 

active  immunization  of  animals  is  also  frequently  undertaken 

with  the  view  of  obtaining  sera  for  diagnostic  and  therapeutic  indications. 

In  the  manufacture  of  serum  on  a  larger  scale,  the  horse  is  the  animal 
used  almost  exclusively.  Occasionally  cows,  sheep,  donkeys  or  mules  are 
selected.  In  small  laboratories  usually  rabbits,  guinea-pigs,  white  mice, 
rats,  and  only  occasionally  goats  or  sheep  are  employed. 

The  process  of  immunization  evokes  a  marked  disturbance  in  the  general 
health  of  the  animals.  For  this  reason  they  must  be  well  kept  in  warm 
places,  and  well  fed  with  nutritious  food.  As  far  as  their  power  of  producing 
antibodies  is  concerned,  there  are  individual  differences  even  among  the 
same  species  of  animals;  thus  if  five  horses  are  immunized  against  diphtheria, 
some  will  give  much  better  curative  sera  than  the  others.  In  general, 
the  younger  animals  are  preferable. 

Any  substance  which,  when  injected  into  an  organism,  can  stimulate  the 
production  or  formation  of  an  antibody,  has  been  conveniently  termed  "  anti- 
gen." After  the  injection  of  such  an  antigen,  special  notice  should  be  taken 
of  the  animal  in  reference  to  temperature,  weight,  the  excitation  of  diarrhea 
or  the  occurrence  of  abscesses,  infiltrates,  edema  or  paralysis. 

If  an  animal  dies,  a  careful  postmortem,  and  if  possible,  a  bacteriological 
examination  should  be  made.  It  should  be  the  aim  to  ascertain  if  death  was 
induced  by  the  inoculated  antigen,  by  contamination  or  secondary  infection. 
One  should  always  keep  in  mind  the  possibility  of  some  of  the  animal 
epidemic  diseases. 

Epidemic  diseases  occurring  in  rabbits  are: 

Animal        i.  RABBIT  SEPSIS. — Presents  itself  in  the  form  of  bronchopneumonia  and 
Infections,      marked  nasal  catarrh.     It  is  very  infectious.     Sick  animals  should  at 
once  be  isolated  or  killed  and  their  cages  thoroughly  disinfected. 
21 


22  ACTIVE    IMMUNIZATION. 

2.  COCCIDIOSIS  gives  changes  in  the  liver  due  to  the  settling  of  the  coccidi  ova  forms. 
The  parasites  are  easily  recognized,  with  the  microscope,  as  present  in  the  pus. 
Following  labor,  guinea-pigs  are  very  susceptible  to  sepsis. 
IN  RATS,  trypanosomiasis  is  of  frequent  existence,  but  is  not  pathogenic. 

The  antigens  are   injected  either  subcutaneously ,  intraperito- 
The  Tech-    neaMy  or   intravenously.     Only  on    exceptional   occasions    is 
niqueof  Active  another  entrance  path  chosen. 

Immuniza-    As  regards  the  amount  to  be  injected,  one  cannot,  very  well, 
give   general   rules.     It   is   important  to  prevent  severe   re- 
actions, although  the  question  is  still  a  disputed  one,  whether 
marked  reactions  tend  to  produce  a  better  immunity.     It  is  certain,  how- 
ever, that  inoculations  of  antigens  in  such  minute  doses  as  to  apparently 
give  no  reaction,  can  still  lead  to  immunity  and  the  production  of  antibodies. 
Occasionally  a  single  injection  suffices  for  immunization.     Repeated  in- 
oculations are  usually  necessary,  especially  so  when  a  "highly  valent"  serum 
is  desired,  i.e.,  one  containing  a  great  number  of  antibodies  or  having  high 
protective  properties. 

When  repeated  inoculations  are  undertaken,  there  are  various  methods  of 
procedure. 

i a.  A  small  dose  of  antigen  is  injected.  If  a  reaction  sets  in,  one  waits 
until  this  reaction  has  entirely  subsided,  then  (not  before  the  fifth  day)  the 
second  injection — a  somewhat  larger  dose — is  given.  After  an  interval  of 
'  5  to  8  days,  a  third  injection  of  a  still  higher  dosage  is  administered,  and 
so  on,  again. 

i  b.  The  intervals  are  the  same,  but  the  amounts  of  antigen  remain  the 
same  at  each  injection. 

Both  of  these  methods  give  excellent  results  and  therefore  are  most 
frequently  used. 

2.  For  several  successive  days,  a  small  or  medium  dose  of  antigen  is 
injected.     Each  injection  produces  only  a  slight  reaction. 

This  last  scheme  according  to  Fornet  is  especially  suitable  for  the  gain- 
ing of  precipitation  sera.  As  is  evident,  it  has  the  advantage  of  gaining 
the  immunity  rapidly. 

3.  Inoculations  are  given  at  very  long  intervals  (intermissions  of  four 
weeks  or  more).     This  method  produces  good  sera,  but  has  the  disadvan- 
tage of  requiring  too  long  a  time. 

The  methods  of  active  immunization  can  also  be  divided  according  to 
the  nature  of  the  antigen. 

1.  Immunization  with  a  living  virus, 

2.  Immunization  with  a  dead  virus, 

3.  Immunization  with  bacterial  extracts, 

4.  Immunization  with  bacterial  toxins. 


CLASSIFICATION    OF    BACTERIA.  23 

i.  Immunization  with  a  Living  Virus. 

This  method  of  immunization  simulates  most  closely  the  immunity 
attained  spontaneously  in  overcoming  an  infection.  Although  this  im- 
munity is  very  strong,  and  lasts  for  a  long  period  of  time,  its  disadvantages 
lie  in  that  it  is  attained  with  difficulty;  frequently  the  dose  of  virus  injected 
causes  serious  symptoms  of  infection.  Various  procedures  have  therefore 
been  advocated  to  so  diminish  the  toxicity  of  the  immunizing  agent  that  only 
immunization  effects,  anoT^no  toxic  symptoms  be  obtained.  This  was 
attempted  either  by  the  reduction  of  the  number  of  organisms  employed,  so 
that  very  minute  doses  wrere  inoculated,  or  by  the  diminution  of  the  infec- 
tious nature  of  these  bacteria  (virulence  so  called) . 

The  first  method,  however,  was  *not  found  applicable  to  all  ca-ses.  The 
infectious  nature  of  the  different  bacteria  varies  markedly.  The  same 
bacterium  reacts  differently  with  different  animals.  While  some  animals 
possess  a  natural  immunity  against  certain  bacteria,  others  exhibit  a  dis- 
tinct susceptibility  to  the  same  micro-organisms.  The  conceptions  there- 
fore of  pathogenicity  and  virulence  are  purely  of  a  relative  nature.  In  talking 
of  the  pathogenicity  of  bacteria,  one  should  always  mention  the  class  of  animal 
for  which  these  bacterii  are  pathogenic. 

Bail  has  used  this  principle  of  pathogenicity  in  classifying  bacteria.     He 
Bail's  Classi-  mentions  the  following  three  classes: 
fication  of      a.  Saprophytes. 

Bacteria.       b.  Half  or  partial  parasites. 
c.   Whole  or  pure  parasites. 

To  the  class  of  saprophytes  belong  all  those  bacteria  which  when  injected  even  in 
larger  doses  do  not  produce  any  characteristic  disease;  these  are  also  known  as  apatho- 
genic — e.g.,  hen  cholera  bacilli  for  human  beings. 

Classed  as  half  parasites  are  those  bacteria,  according  to  Bail,  the  infectious  nature 
of  which  depends  upon  the  quantity  of  bacteria  injected.  While  the  injection  of  a 
rabbit  with  i/iooo  of  a  loopful  of  a  typhoid  culture  will  produce  no  evidences  of  disease, 
one-tenth  of  a  loopful  will  result  in  slight  increase  in  temperature,  loss  of  appetite,  and 
eventually  a  local  redness  at  the  site  of  the  injection.  One  loopful  may  bring  about 
the  death  of  the  animal.  The  manifestations  are  dependent  entirely  upon  the  number 
of  bacteria  injected.  The  smaller  the  number,  the  milder  the  symptoms,  until  one 
reaches  the  stage  below  which  no  disturbances  at  all  are  visible. 

Pure  parasites  are  those  which  have  no  subletal  dose.  Even  the  smallest  amount, 
when  injected,  will  produce  death.  As  examples,  the  tubercle  bacillus  for  guinea-pigs, 
and  bacilli  belonging  to  the  group  of  Hemorrhagic  Septicemia  for  rabbits.  Of  the  last 
mentioned  1/10,000,000,000  of  a  loopful  of  some  cultures  kills  a  rabbit  within  twenty- 
four  hours  with  the  symptoms  of  a  septicemia;  in  other  words,  the  injection  of  i  c.c.  of  a 
dilution  of  one  loopful  of  culture  in  ten  million  liters  of  water  suffices  to  kill  the  rabbit. 
Furthermore,  the  number  of  bacteria  increases  so  greatly  in  the  body  of  the  rabbit  that 
numerous  bacteria  can  be  demonstrated  in  every  drop  of  blood  and  in  all  organs  and 
body  fluids. 

The  same  organism  is  a  saprophyte  for  the  human  being  and  a  half  parasite  for  the 
guinea-pig  if  injected  subcutaneously  and  a  complete  parasite  by  intraperitoneal  injection. 
The  conceptions  therefore  of  complete  or  partial  parasite  as  well  as  of  saprophyte  are  only 
relative  and  are  dependent  upon  the  bacteria,  the  animal  species,  and  the  mode  of  infection. 


24  *  ACTIVE   IMMUNIZATION. 

It  is  now  clear  that  immunization  with  living  bacteria  can 

Example     on^  ^e  undertaken  if  the  latter  belong  to  the  class  of  half- 

of  Active     parasites.     Pure  parasites  are  excluded  from  this  method. 

Immunization  As  an  example  of  such  procedure  can  be  given  the  immu- 

with  Living   nization  of  a  guinea-pig  by  intraperitoneal  injections  with 

acilh.       living    typhoid    bacilli.     Preliminary    to    this,    the   virulence 

of  the  typhoid  culture  must  be  ascertained. 

a.  Preliminary  test  to  titrate  the  virulence  of  the  typhoid  culture. 

1.  Guinea-pig  i./I.  1909  1/20  loopful  of  typhoid  culture  intraperitoneal. 

2./I.  active. 

8./I.  alive. 

2.  Guinea-pig  i./I.  1909  i/io  loopful  of  typhoid  culture  intraperitoneal. 

2./I.  active. 

8./I.  alive. 

3.  Guinea-pig  i./I.  1909  1/8  loopful  of  typhoid  culture  intraperitoneal. 

2. /I.  slightly  sick,  does  not  eat. 

3. /I.  active. 

8./I.  alive. 

4.  Guinea-pig  i./I.  1909  1/6  loopful  of  typhoid  culture  intraperitoneal. 

2. /I.  sick,  does  not  eat,  hair  raised. 

3./I.  still  sick. 

4./I.  more  active. 

8./I.  alive. 

5.  Guinea-pig  i./I.  1909  1/5  loopful  of  typhoid  culture  intraperitoneal. 

2. /I.  sick,  does  not  eat,  hair  raised. 

3./I.  very  weak,  when  placed  on  side  remains  so. 

4-/I.  t    ' 

6.  Guinea-pig  i./I.  1909  1/6  loopful  of  typhoid  culture  intraperitoneal. 

2./I.       '  f 

From  this  experiment  it  becomes  evident  that  the  letal  dose  of  this  particular  strain 
of  typhoid  culture  is  1/4  to  1/5  of  a  loopful  for  guinea-pigs  by  intraperitoneal  injection. 
Immunization  therefore  must  be  started  with  a  smaller  dose  — e.  g.,  i/io  of  a  loopful. 

b.  Immunization. 

1.  Guinea-pig      8./I.  1909  i/io  loopful  of  typhoid  culture  intraperitoneal. 

i6./I.  1/8  loopful  of  typhoid  culture  intraperitoneal. 

22. /I.  1/4  loopful  of  typhoid  culture  intraperitoneal. 

Animal  remains  active  and  healthy. 
30./I.  I  loopful  of  typhoid  culture  intraperitoneal. 

5./II.  2  loopfuls  of  typhoid  culture  intraperitoneal. 

Animal  remains  active  and  healthy. 

2.  Guinea-pig      8./I.  1909  i/io  loopful  of  typhoid  culture  intraperitoneal. 

i6./I.  1/4  loopful  of  typhoid  culture  intraperitoneal. 

Animal  remains  active  and  healthy. 

3.  Guinea-pig      8./I.  1909  i/io  loopful  of  typhoid  culture  intraperitoneal. 

i6./I.  2  loopfuls  of  typhoid  culture  intraperitoneal. 

I7-/I.  animal  is  sick  and  does  not  eat. 

i8./I.  animal  is  very  weak. 

i9./L  t 


VACCINATION  AGAINST   SMALL-POX.  25 

"ontrol  animals  always  die  within  twenty-four  hours,  as  in  previous  experiment,  on 
injection  of  1/4  of  a  loopful. 

From  experiment  with  guinea-pig  i,  it  can  be  learned,  that  by  gradual  increase  of 
the  immunizing  dose,  a  state  of  immunity  is  reached  which  can  overcome  an  infection 
produced  by  a  high  multiple  of  the  dosis  letalis. 

Experiment  2  and  3  prove  that  even  a  single  preliminary  injection  suffices  to  prevent 
the  death  of  an  animal  upon  subsequent  receipt  of  the  lethal  dose  of  the  same  bacteria; 
but  that  this  single  inoculation  is  not  sufficient  to  prepare  the  organism  against  a  very 
severe  future  infection.  The  attained  immunity  is  therefore  only  relative,  not  absolute. 

Analogously  it  is  possible  to  immunize  by  subcutaneous  and  intra- 
venous injections.  The  latter  method  is  usually  the  one  of  choice  when 
half  parasites  are  employed,  as  the  highest  and  quickest  grade  of  immunity 
is  thus  reached.  It  carries  with  it,  however,  the  greatest  danger  and  fre- 
quently results  in  death  to  the  animal. 

The  method  of  immunization  with  small  doses  of  living,  fully  virulent 
bacteria,  has  thus  far  been  made  use  of  only  in  animals.  In  man  this 
experience  has  not  been  carried  into  effect.  It  is  feared  that  the  bacteria  may 
increase  very  rapidly  and  give  rise  to  severe  disturbances.  The  method  has 
therefore  been  altered  and  instead  of  using  virulent  material  for  immu- 
nization, only  a  weakly  infectious  or  attenuated  virus  is  employed. 

Vaccination  against  Small-pox. 

This  is  the  best  known  example  of  active  prophylactic 
immunization.     To  Jenner  belongs  the  credit  of  having  been 
the  first  one  to  apply  this  principle.     Vaccination  against 
small-pox  consists  in  inoculation  of  an  attenuated  form  of 
small-pox  germs,  the  diminution  in  virulence  being  brought  about  by  pass- 
age through  the  body  of  a  calf,  a  less  susceptible  animal  than  man.     The 
vesicles  formed  on  the  vaccinated  person  contain  these  attenuated  germs. 
This  lymph  can  be  used  for  the  inoculation  of  other  individuals,  as  the 
germs  do  not  regain  their  virulence  by  repassage  through  man. 

Inasmuch  as  it  is  not  within  the  scope  of  this  book  to  go  into  the  details 
of  the  preparation  of  the  lymph  or  the  technique  of  vaccination,  a  brief 
survey  of  the  benefits  of  vaccination  will  amply  suffice  and  this  may  be  seen 
from  the  table  hereunto  appended. 

The  mortality  from  small-pox  per  100,000  population  was  in  the  year 

1862-1876    1882-1896 

in  Prussia  and  Bavaria. 51.6  0.7 

in  Austria 75.2  38.6 

in  Belgium 79. 5  18. 2 

in  England 25.3  2.9 

in  Sweden 26.9  0.5 

This  method  of  immunizing  against  a  virulent  virus  by  inoculating  with  an 
attenuated  form  of  the  same,  is  known  as  Jennerization.  Pasteur  recognized 
that  this  method  had  general  application  and  similarly  used  attenuated  but 


26  ACTIVE    IMMUNIZATION. 

still  living  cultures,  "vaccins"  so-called,  to  immunize  against  hen  cholera, 
swine  plague,  and  anthrax.  The  same  principle  underlies  Pasteur's  antirabic 
vaccination. 

Antirabic  Vaccination. 

In  all  civilized  countries  their  exist  at  present,  special  institutions, 
either  directly  under  the  city  control  or  appointed  by  the  city,  where  the 
Pasteur  treatment  for  rabies  is  conducted.  It  is  the  duty  of  the  general 
practitioner,  on  getting  a  suspicious  case  of  rabies  to  advise  his  patient  to 
undergo  this  special  therapy  and  to  send  the  rabid  animal,  its  head  or  brain 
preserved  in  glycerin,  to  the  institute  as  soon  as  possible  for  the  purpose  of 
ascertaining  the  presence  of  rabies.  Up  to  the  present  the  actual  cause 
of  hydrophobia  is  unknown.  Most  recently  Negri  has  described  parasites, 
known  as  Negri  bodies,  in  the  large  nerve  cells  of  the  cerebral 


cortex>  cerebellum,  etc.     Pasteur  found,  that  rabies  can  be 


Treatment     transmitted  to  dogs  by  injecting  them  subdurally  with  the 
brain  substance  of  rabid  animals.     This  ordinary  virus  con- 
taining material  is  known  as  Street  Virus. 

The  incubation  period  of  rabies  is  very  long.  It  varies  from  about  three 
weeks,  to  [possibly],  some  years.  By  passing  the  virus  through  monkeys, 
the  incubation  period  is  considerably  increased.  After  the  successive 
passage  through  five  or  six  animals,  the  virus  becomes  so  weakened  that 
infection  is  almost  impossible.  Reversely,  increase  of  the  virulence  may 
be  affected  by  passing  the  virus  through  a  successive  number  of  rabbits 
which  are  very  sensitive  to  the  disease.  After  passage  through  a  large 
number  of  such  animals,  the  incubation  period  is  gradually  shortened  from 
about  three  weeks  or  a  little  less  to  a  constant  period  of  six  or  seven  days. 
Further  diminution  in  the  period  of  incubation  was  impossible  and  there- 
fore Pasteur  called  this  "  Virus  fixe"  His  first  experiments  in  immunization 
were  made  by  passing  the  weakened  monkey  virus  through  rabbits  and  then 
treating  dogs  with  the  spinal  cords  of  the  latter. 

Later  on,  Pasteur  discovered  that  instead  of  passing  the  virus  through 
monkeys,  he  could  diminish  its  virulence  by  drying  the  spinal  cords  derived 
from  rabid  animals,  for  varying  periods  of  time.  In  this  way  he  could 
prepare  an  entire  series  of  graduated  strengths.  The  material  used  for 
this  drying  was  not  the  street  virus,  but  that  obtained  by  successive  passage 
through  rabbits  or  "virus  fixe"  which  possessed  very  constant  immunizing 
and  infectious  properties.  By  drying  the  "virus  fixe"  over  caustic  potash 
at  a  temperature  of  23°  to  25°  C.  for  five  days,  its  regular  incubation  period 
of  7  days  was  very  much  prolonged.  Increase  in  the  length  of  drying 
caused  the  entire  loss  of  virulence  in  the  spinal  cord. 

Pasteur  immunized  dogs  as  follows:  He  began  with  the  injection  of  a  virulent 
spinal  cord  which  had  been  dried  for  thirteen  days  and  every  following  day  injected 


TECHNIQUE    OF   ANTIRABIC    VACCINATION. 


subcutaneously  some  fresher  spinal  cord,  i.e.  (dried  for  a  lesser  period  of  time),  until 
finally  he  used  virus  dried  only  for  one  day.  The  animals  thus  treated  were  immune 
against  the  bites  of  rabid  dogs  as  well  as  subdural,  subcutaneous,  and  intravenous 
infection  with  "virus  fixe"  and  street  virus.  This  procedure  was  strongly  recommended 
by  Pasteur,  who  brilliantly  contributed  the  observation,  that  if  an  animal  was  infected 
but  did  not  as  yet  show  symptoms,  these  could  be  prevented  by  a  similar  modus 
operandi,  as  above  mentioned. 

In  man,  the  inoculation  is  carried  out  on  the  same  principle.  The 
fact  that  the  incubation  period  of  hydrophobia  is  very  long,  makes  the  pro- 
phylactic inoculations  of  greater  service.  Only  rarely  is  this  period  less 
than  six  weeks,  usually  considerably  longer — up  to  584  days,  entirely  depend- 
ent upon  the  virulence  of  the  virus  and  the  point  of  infection. 

Technique  of  Antirabic  Vaccination  in  Man. 

The  actual  vaccine  consists  of  i  c.c.  (2-3  mm.  length)  of  the  substance  of 
the  spinal  cord  of  a  rabbit  which  has  been  killed  by  inoculation  with  the 
fixed  virus,  rubbed  up  into  a  fine  emulsion  with  5  c.c.  of  sterile  0.85  NaCl 
solution.  About  i  to  3  c.c.  of  the  resulting  fluid  are  injected  subcutaneously 
into  the  skin  of  the  abdomen.  A  cord  dried  for  fourteen  days  is  used  for 
the  first  injection,  emulsions  of  less  attenuated  virus  are  used  on  succeeding 
occasions  until  finally  a  portion  of  a  spinal  cord  dried  for  only  three  or  four 
days  is  employed.  Pasteur's  schemes  of  the  actual  doses  can  thus  be  drawn 
up. 

a.  For  infections  at  points  distant  from  the  central  nervous  system  (mild  infections}. 


Day  of  injection 

i 

2 

3 

4 

5 

6 

' 

8 

9 

10 

II 

12 

13 

14 

15 

Number   of    days 

14 

12 

10 

8 

6 

5 

5 

4 

3 

5 

5 

4 

4 

3 

3 

cord  was  dried. 

+ 

+ 

+ 

+ 

+ 

13 

II 

9 

7 

6 

1 

Amount  injected  .  . 

3-o 

3-o 

3-o 

3.0      2.0      I  .0 

I  .0 

r  .0    i  .0 

2  .0 

2.0 

2  .O 

2.0 

2.0 

2.0 

b.  For  head  wounds  (severer  infections}. 


Day  of  injection  .  . 

i 

2 

2 

4 

5 

6 

7 

8 

Number  of  days 

14                  12 

10               8 

6  +  6 

5 

5 

4 

3 

4 

cord  was  dried. 

+                    + 

+            + 

I3                   II 

9              7 

In  A.  M.    In  P.  M. 

In  A.  M.    In  P.  M. 

Amount  injected. 

A.  M.          P.  M. 

2  C.C.  A.  M.    2  C.C.  P.  M. 

A.  M.          P.  M. 

2  . 

2  . 

2. 

i  . 

2  . 

28 


ACTIVE   IMMUNIZATION. 


Day  of  injection.  . 

10 

II 

12 

J3 

14 

15 

16 

17 

18 

19 

20 

21 

Number  of  days 

5 

5 

4 

4 

3 

3 

5 

4 

3 

5 

4 

3 

cord  was  dried. 

Amount  injected.. 

2  . 

2  . 

2  . 

2. 

2  . 

2  . 

2  . 

2  . 

2. 

2  . 

2  . 

2  . 

The  drawback  to  this  classical  method  of  Pasteur,  consists  in  using  the 
virulent  material  rather  late  in  the  course  of  the  inoculations.  A  more 
energetic  treatment  has  therefore  been  advised.  There  is  no  added  danger 
in  doing  this  because  the  virus  fixe  in  contrast  to  the  street  virus  is  not  at  all 
or  only  slightly  infectious  for  man. 

Hogyes  in  Buda  Pesth  uses  the  virus  fixe  right  from  the  start.  He  begins 
with  marked  dilutions  (1/10,000)  and  gradually  increases  them  to  i/ioo. 
The  theory  underlying  this  procedure  is,  that  the  usual  method  of  attenu- 
ation by  drying  alters  the  quantity  of  the  virus  but  not  its  quality;  hence  the 
same  result  may  be  obtained  by  simple  dilution. 

Ferran  successfully  employs  the  virulent  virus  in  large  doses  right  from 
the  onset  of  the  treatment.  Especially  in  very  severe  infections,  as  in  bites 
from  wolves,  is  this  procedure  justifiable. 

The  exact  arrangement  of  doses  varies  a  little  at  different  institutions. 
In  Berlin,  it  is  considered  that  the  virulence  of  the  dried  cord  is  lost  on  about 
the  eighth  day  instead  of  the  fourteenth.  Hence  in  the  hydrophobia  depart- 
ment of  the  Berlin  Institute  for  Infectious  Diseases,  the  authorities  have 
adopted  the  following  scheme,  which  stands  midway  between  Pasteur's 
classical  method  and  the  extreme  procedure  of  Ferran. 


Scheme  for  treatment  of  mild  infections : 


Day  of  injection  .... 

i 

2 

3 

4 

5 

6 

7 

8 

9 

10 

II 

12 

J3 

14 

15 

16 

*7 

18 

19 

2O 

21 

Number   of   days 

8-7-6 

5-4 

4-3 

5 

4 

3 

3 

2 

2 

5 

5 

4 

4 

3 

3 

2 

2 

4 

3 

2 

2 

cord  was  dried. 

Amount  injected  in 

0.5  of 

i  .5  of 

2  .O 

3-° 

3- 

i-5 

2 

I 

I 

2 

2 

2 

2 

2 

2 

i-5 

!-5 

2 

2 

I'S 

2 

cubic   centimeters 

each 

each 

I  .O 

of  an  emulsion  i 

c.c.  of  cord  in  5c.c. 

of  sterile  bouillon. 

VACCINATION  AGAINST   TUBERCULOSIS. 

Scheme  for  treatment  of  severe  infections. 


29 


Day  of  injection  

i 

2 

3 

4 

5 

6 

7 

8 

9 

10 

II 

12 

J3 

14 

15 

16 

17 

18 

19 

2O 

Age  of  cord  

8-7-6 

4-3 
i-5 

5-4 
i-5 

3 

3 

2 

2 

i 

5 

4 

4 

3 

3 

2 

2 

4 

3 

2 

2 

3 

Amount.  . 

i-5 

2 

2 

I 

I 

i 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

In  severe  injuries  the  entire  treatment  is  repeated  after  one  month's  interval. 

There  is  at  present  no  doubt  whatsoever  as  to  the  value  of  these  antirabic  vaccinations. 
While  the  mortality,  compiled  from  a  great  number  of  statistics,  of  the  untreated  cases, 
of  those  infected  or  exposed  to  infection  is  15  to  16  per  cent.,  the  death  rate  of  those 
treated  at  the  Berlin  Institute  during  1898  to  1901  was  0.55  per  cent.  Similar  figures 
are  given  by  the  other  institutions. 

Attempts  have  been  made  to  employ  this  principle  of  virus  attenuation 

for  other  infections.     Behring  and  Koch  tried  immunization 

Vaccination    against  bovine  tuberculosis  by  inoculation  with  living  human 

Against       tubercle  bacilli.     The  material  used  for  the  above  inocula  ions 

Tuberculosis.  can  be  bought  under  the  name  of  Bovovaccine  (v.  Behring) 

and  Tauruman  (Koch) . 

Tauruman  is  prepared  by  the  Hochst  Farbwerke  and  is  put  up  in  sealed  glass 
tubes  which  contain  0.02  to  0.04  gm.  of  living  tubercle  bacilli  suspended  in  10  c.c. 
of  normal  saline  solution.  This  Tauruman  is  previously  examined  in  Ehrlich's  Institute 
and  note  is  taken  of  its  purity,  quantity  of  bacteria,  virulence  against  guinea-pigs  and 
avirulence  against  rabbits  (characteristics  of  the  human  type  of  tubercle  bacilli). 

To  this  class  of  experimental  work  belong  also  the  attempts  of  Friedmann 
to  immunize  against  human  tuberculosis  by  the  use  of  the  tubercle  bacilli 
of  cold  blooded  animals,  and  those  of  Wassermann,  Ostertag  and  the  author, 
to  inoculate  against  hog  cholera  with  living  cultures  of  mouse  typhoid. 

Besides  the  preceding  way  of  virus  attenuation  by  passage  through 

animals,  there  are  other  methods  employed  for  the  diminu- 

Other          tion.  of  the  toxicity  of  the  virus.     Growing  the  bacteria  ^at 

Methods  of    too  high  a  temperature,  or  exposing  bacterial  emulsions  to 

Vaccine      light,   disinfectants   or   moderate   heating,   accomplishes   the 

Preparation.    same  purpOse. 

The  mixture  of  bacteria  with  their  specific  serum  (i.e.,  serum 
obtained  from  animals  that  have  been  inoculated  with  these  bacteria), 
also  diminishes  the  strength  of  the  inoculated  bacteria.  Such  bacteria  are 
designated  by  Bordet  as  "sensitized"  bacteria.  By  allowing  this  mixture 
to  remain  for  some  time,  the  bacteria  attach  their  specific  antibodies  so  that 
after  centrifugalization  the  added  specific  serum,  now  devoid  of  its  specific 
antibodies  is  removed,  and  the  sensitized  bacteria  can  be  used  as  vaccines. 
Inoculations  of  the  latter  rarely  produce  any  infiltration.  The  same  object 


30  ACTIVE    IMMUNIZATION. 

can  also  be  accomplished  by  injecting  bacteria  and  at  the  same  time  also 
their  specific  serum.  This  is  technically  simple  and  is  known  as  the  "Simul- 
taneous Method"  It  has  shown  itself  to  be  of  great  value  in  Lorenze's  pro- 
phylactic inoculations  against  swine  erysipelas. 

2.  Immunization  with  Dead  Bacteria. — Immunization  with  dead  bac- 
teria was  first  performed  by  Toussaint,  Salmon  and  Smith,  and  Chamber- 
land  and  Roux. 

This  method  is  to  be  distinctly  separated  from  those  already  discussed.  Bail  claims 
that  the  immunization  with  living  bacteria  as  well  as  by  aggressins  (to  be  mentioned  later) 
is  an  immunization  against  the  infectious  disease;  while  the  immunization  with  dead 
bacteria  is  an  immunization  against  the  bacterial  bodies.  While  this  holds  true  for 
some  bacteria,  it  is,  to  say  the  least,  questionable  whether  it  can  be  considered  as  a 
general  rule. 

Whenever  a  real  immunity  is  desired — that  is,  protection  against  disease, 
a  vaccine  either  in  the  form  of  living  or  attenuated  bacteria  should  be  given 
the  preference.  Up  to  a  certain  degree  the  extracts  of  living  bacteria,  and 
the  natural  and  artificial  aggressins  can  be  similarly  employed.  If,  however, 
no  real  immunity,  but  just  a  serum  containing  a  great  number  of  antibodies 
is  wanted,  as  in  serum  diagnosis,  for  agglutination,  bacteriolysis,  complement 
fixat  on,  etc.,  then  immunization  by  dead  bacteria  is  just  as,  if  not  more  so, 
efficient. 

Recently,  the  question  has  been  raised  whether  the  antibodies  produced 
by  immunization  with  heated  antigens  are  identical  with  those  obtained 
with  unheated  antigens.  The  experiments  of  Obermeyer  and  Pick  which 
will  be  referred  to  under  proteid  immunization,  seem  to  prove  that  they 
are  not  alike.  For  laboratory  work  it  is  advisable  to  use  living  cultures 
only  in  cases  of  absolute  necessity. 

In  heating  bacteria  to  destroy  their  virulence  and  thus  be  suitable  for 
inoculation,  we  must  be  very  careful  not  to  raise  the  temper- 
Death  of     ature  to  such  a  degree  where  not  only  the  toxicity  but  also  the 
Bacteria      immunization  power  is  destroyed.     It  is  best  to  employ  the 
by  Heat,      minimum  amount  of  heat  which  will  kill  the  respective  bacteria. 
For  most  of  these  as  Typhoid,  Paratyphoid,  Colon,  and  Dysen- 
tery bacilli,  Cholera  Vibrios,  Meningo-,  Staphylo-,  Strepto-  and  Pneumo- 
coccis,  one  hour  at  60°  C.  is  sufficient. 

The  bacteria  are  grown  upon  agar  cultures  and  the  required  amount 
is  removed  and  suspended  in  sterile  physiological  salt  solution  or  bouillon. 
This  suspension  is  then  placed  into  a  hot  water  bath  or  thermostat  regulated 
at  60°,  for  one  hour. 

If  the  bacteria  employed  are  highly  infectious,  one  must  be  sure  that  all 
bacteria  have  been  killed.  This  must  especially  be  noted  when  giving 
prophylactic  inoculations  in  man.  Several  drops  of  the  emulsion  are  there- 


PROPHYLACTIC  TYPHOID  INOCULATION.  31 

fore  subplanted  on  agar  tubes  and  incubated  for  a  day  or  two.  If  a  growth 
appears,  the  emulsion  is  to  be  reheated;  if  not  it  can  be  considered  sterile. 

The  mode  of  immunization  is  the  same  as  has  been  described  for  the 
living  bacteria.  In  general  the  dosage  to  be  used  may  be  larger. 

Small  doses  are  injected  at  first,  followed  later  on  by  increasing  quanti- 
ties at  intervals  of  five  to  eight  days.  e.g. 

Intravenous  inoculation  of  a  rabbit  with  dead  typhoid  bacilli. 

Result. — Protection  against  living  virulent  bacteria,  appearance  of 
agglutinins,  bacteriolysins,  bacteriotropins  and  complement  binding  sub- 
stances in  the  serum. 

i. /I.  1909.  Rabbit  No.  I.  i  loopful  of  a  typhoid  agar  slant  culture  killed  at  60°  and 

injected  intravenously. 

6./I.  4  loopfuls  of  typhoid  culture  killed  at  60°  and  injected  intra- 

venously. 

12. /I.  i  culture  of  typhoid  killed  at  60°  and  injected  intravenously. 

20. /I.  Infection  with  i  culture  of  the  living  typhoid  bacilli  injected 

intravenously.     Animal  remains  alive. 
Rabbit  No.  2.     Control. 

20. /I.  Infection:  1/4  loopful  of  living  typhoid  bacteria  intravenously. 

22. /I.  f  (death). 

The  use  of  killed  typhoid  bacteria  for  prophylactic  immunization  has 
recently  been  widely  adopted.  This  has  been  stimulated  to  a  great  degree 
by  the  successful  experiments  of  Wright,  and  PfeifTer  and  Kolle. 

Wright's  Method  of  Prophylactic  Typhoid  Inoculation. 

The  vaccine  or'ginally  employed  by  Wright  for  these  inoculations  con- 
sisted of  highly  virulent  cultures  of  Bacillus  Tpyhosus  grown  in  broth  for 
twenty-four  to  forty-eight  hours  (sometimes  even  for  four  weeks),  and 
sterilized  by  heating  at  60°  C.  The  vaccine  was  then  standardized,  i.e., 
the  strength  of  the  vaccine  was  fixed  in  accordance  with  another  of  known 
strength,  the  dosage  of  which  had  been  gauged  by  inoculations  in  man. 

The  early  form  of  standardization  consisted  in  determining  the  toxicity 
of  the  virus.  Guinea-pigs  weigh.'ng  250  to  300  gm.  were  inoculated  sub- 
cutaneously  with  0.5,  0.75,  i.o  and  1.5  c.c.  of  the  vaccine  respectively. 
Death  to  some  of  the  animals  would  come  in  twelve  hours  to  three  days. 
The  amount  required  to  kill  a  guinea-pig  weighing  100  grammes  or  rather 
the  proportional  fraction  of  the  dose  which  proved  fatal  to  the  one  of  250  to 
300  gm.  was  taken  as  the  standard  dose  for  injection  in  man.  Wright  sub- 
sequently found  that  better  results  were  obtained,  if  the  vaccine  was  pre- 
pared from  twenty-four  hour  cultures  grown  upon  the  surface  of  agar,  and 
after  emulsification,  standardized  so  as  to  contain  1,000  millions  of  typhoid 
bacilli  in  every  cubic  centimeter.  This  method  of  standardization,  the 
details  of  which  will  be  given  in  the  chapter  on  Opsonins,  is  effected  by 


32  ACTIVE   IMMUNIZATION. 

counting  the  number  of  bacteria  under  the  microscope.  At  the  first  inocu- 
lation, the  patient  received  750  to  1000  million  of  these  dead  bacteria  and  at 
the  second,  eleven  days  later  double  the  first  dose  injected. 

Local  and  general  reactions  follow  the  inoculations.  Thus  local  redness 
and  swelling  of  the  skin,  lymphangitis  and  enlargement  of  the  neighboring 
glands  are  the  usual  consequences.  The  inflammation  can  at  times  be 
severe  enough  to  simulate  erysipelas.  The  general  symptoms,  on  the  other 
hand,  may  consist  of  a  general  feeling  of  illness,  headache,  a  little  fever,  and 
occasionally  nausea,  not  infrequently  accompanied  by  vomiting.  These 
signs  of  indisposition,  however,  pass  off  rapidly  without  leaving  any  perma- 
nent ill  effects.  Six  to  eleven  days  after  the  injection,  an  increase  in  the 
number  of  agglutinating,  bacteriolytic  and  bacteriotropic  bodies  can  be 
demonstrated  in  the  blood  of  the  inoculated  individual. 

As  to  the  effects  of  these  inoculations  opinion  is  somewhat  divided. 
According  to  Wright's  statistics  infections  have  been  diminished  by  about 
one-half,  and  in  single  series  to  one-sixth  or  even  one-twenty-eighth  of  the 
former,  or  control  number.  The  mortality  too  is  much  lower.  Out  of 
1758  individuals  who  had  been  vaccinated,  only  142  or  8  per  cent,  died; 
out  of  10,980  who  had  not  been,  1800  or  16.6  per  cent,  met  death.  From 
numbers  such  as  these,  Wright  has  come  to  the  opinions  he  holds,  and  he 
moreover  believes  that  the  period  of  time  during  which  prophylactic  immu- 
nity can  be  maintained  is  from  two  to  three  years. 

Pfeiffer-Kolle's  Experiments. 

Pfeifler  and  Kolle  prepare  their  vaccine  by  growing  typhoid  bacilli  on 
agar  cultures  and  suspending  a  twenty-four  hours  growth  in  physiological 
NaCl  solution.  The  normal  platinum  loop  is  the  unit  of  standardization. 
A  full  grown  agar  culture  is  considered  as  10  normal  loops  and  as  such  it  is 
diluted  in  4.5  c.c.  of  saline.  This  emulsion  is  placed  into  a  thermostat 
at  60°  for  two  hours  and  then  tested  for  its  sterility.  Sufficient  5  per  cent, 
phenol  solution  is  next  added  to  the  suspension  to  make  up  the  contents 
to  a  0.5  per  cent,  carbolic  solution,  and  the  final  emulsion  is  again  heated  at 
60°  C.  for  thirty  minutes.  One  c.c.  of  the  vaccine  is  thus  equivalent  to  two 
normal  loops  of  culture.  The  amounts  of  vaccine  to  be  injected  have  not 
yet  been  definitely  decided  upon.  The  best  dosage  so  far  is  the  following: 

For  the  ist  injection:  0.3  c.c.  of  the  vaccine. 
For  the  20!  injection:  0.8  c.c.  of  the  vaccine. 
For  the  3d  injection:  i  .o  c.c.  of  the  vaccine. 

The  injection  is  made  subcutaneously  between  the  breast  and  clavicle. 

The  local  and  general  reactions  are  the  same  as  those  observed  with 
Wright's  method.  As  a  result  of  the  injection  only  increased  agglutinins 


PFEIFFER-KOLLE'S  EXPERIMENTS.  33 

and  bacteriolysins,  have  been  found  in  the  blood  serum.     Bacteriotropins 
have  not  as  yet  been  examined  for. 

The  effects  of  these  inoculations  seem  to  be  very  good.  Protection  is  prolonged 
according  to  the  increase  in  the  number  of  injections,  and  if  inoculated  individuals  do 
become  infected,  they  run  a  very  much  milder  course  of  the  disease. 

The  following  statistics  as  given  by  Kuhn  indicate  the  results: 

Inoculated.  Non-Inoculated. 

Very  slightly  ill. . .                                 .186  (50. 13  per  cent.)  331  (36. 55  per  cent.) 

Moderately  ill 96  (25.88  per  cent.)  225  (24. 85  per  cent.) 

Badly  ill 65  (17.52  per  cent.)  234  (25.80  per  cent.) 

Deaths 24  (  6.47  per  cent.)  116  (12.80  per  cent.) 

371  (100      per  cent.)          906  (100      per  cent.) 

The  prophylactic  immunity  according  to  Kuhn  lasts  one  year.     Kolle  has  undertaken 
similar  experiments  against  cholorea. 


CHAPTER  IV. 
ACTIVE  IMMUNIZATION. 

Immunization   with    Bacterial    Extracts. — Aggressin    Experiments. 

The  marked  infectious  nature  of  the  organisms  belonging  to  the  class  of 
"pure  parasites,"  makes  it  very  difficult  to  produce  an  immunity  against 
them.  They  possess  no  sublethal  dose  in  their  living  state,  and  if  used 
when  dead,  will  produce  no  prophylactic  immunity.  Pasteur  therefore, 
by  artificial  attenuation  of  these  living  virulent  bacteria,  had  succeeded,  in 
part,  to  obtain  vaccines  of  several  of  them.  The  methods,  however,  that 
he  employed  were  totally  impracticable,  for  not  infrequently  in  the  use  of  the 
vaccine,  the  disease  which  it  was  the  object  to  prevent,  was  instigated.  It 
was  therefore  a  distinct  and  important  triumph  when  Bail  and  Weil  showed 
that  immunity  against  these  parasites  could  be  attained  by  using  as  vaccine 
antigen,  the  so-called  "aggressms;"  i.e.,  exudates  from  animals  that  had 
been  infected  with  the  respective  bacteria. 

Bail's  explanation  of  the  aggressin-immimization  method  is  entirely  theoretical.  He 
believes  that  during  an  infection,  the  bacteria  secrete  certain  agents  which  counteract 
or  entirely  destroy  the  infected  organism's  protective  powers,  especially  phagocytosis. 
These  bodies  he  called  aggressins  and  they  were  distinguished  by  the  fact  that  they 
were  formed  by  living  bacteria,  and  only  in  the  living  body.  According  to  Bail,  the 
pathogenicity  of  bacteria  depends  upon  their  power  to  produce  these  aggressins.  If 
this  theory  be  correct,  it  should  be  possible  to  demonstrate  aggressins,  especially  in 
infections  where  the  protective  power  of  the  organism  is  almost  nil,  as  for  example  an 
infection  produced  by  the  bacteria  belonging  to  the  group  of  hemorrhagic  septicemia. 
Unfortunately,  in  actual  practice  this  is  not  so. 

The  following  experiment,  however,  gives  an  idea  of  the  true  nature  of 
these  aggressins  and  how  they  are  obtained. 

At  first,  an  infecting  agent — the  bacillus  of  swine  pest,  may  be  chosen. 
This  micro-organism  belongs  to  the  same  class  as  chicken  cholera  and  fowl 
plague,  and  is  distantly  related  to  the  human  pest.  For  rabbits,  this  bacillus 
is  a  pure  parasite,  for  guinea-pigs,  by  subcutaneous  inoculation,  a  half 
parasite. 

The  Obtention  of  Aggressins. 

One  drop  of  a  twenty-four-hour  broth  culture  of  this  swine  pest  bacillus, 
in  5  c.c.  bouillon,  is  injected  intrapleurally  into  a  rabbit  in  the  following 
manner. 

34 


FIRST   FUNDAMENTAL  AGGRESSIN   TEST. 


35 


A  small  incision  is  made  in  one  of  the  intercostal  spaces  on  the  side  of 
the  chest,  and  through  this  wound  a  long  canula  is  introduced  into  the 
pleural  cavity.  Following  the  injection,  the  animal  as  a  rule,  rapidly 
succumbs  to  the  infective  organism.  On  autopsy,  the  pleural  cavity  is 
found  to  contain  an  exudate  of  a  reddish-brown  color  (hemorrhagic)  on 
the  side  where  the  inoculation  was  given,  and  of  yellow  serous  on  the  other 
side.  This  bloody  exudate  measuring  about  15  c.c.  is  removed^  with  a 
sterile  pipette,  placed  into  a  sterile  centrifuge  tube  to  which  is  added  1.5 
c.c.  of  5  per  cent,  carbolic  acid  drop  by  drop  (making  the  entire  solution  a 
1-2  per  cent,  carbolic  acid  dilution) ,  agitated  continually  in  order  to  prevent 
precipitation,  and  followed  by  centrifugalization  at  a  very  high  speed  for 
many  hours  until  it  becomes  very  clear.  The  upper  clear  part  which  is 
now  free  of  bacteria,  or  very  nearly  so,  is  drawn  off  by  a  pipette  and  heated 
for  three  hours  at  44°  C.  Its  sterility  is  then  tested  and  if  no  growth 
appears  after  forty-eight  hours,  it  is  considered  sterile. 

First  Fundamental  Aggressin  Test. 

(Its  power  of  increasing  severity  of  infections.} 


No. 

Animal. 

Date. 

Amount  of  Infective  Material. 

Aggressins. 

Result. 

i 

Guinea-pier. 

6/rV  '05. 

i/ioo  loopful  of  swine  pest 

Remains  alive. 

Jr  o 

subcutaneously. 

2 

Guinea-pig. 

6/IV  '05. 

i/ioo  loopful  of  swine  pest 

+  1.5    c.c. 

f  on  third  day. 

subcutaneously. 

of   aggressins 

subcutane- 

ously. 

7 

Guinea-pig. 

6/IV  '05. 

+  1    S  C  C 

Remains  alive. 

o 

T^   X  .  ^     V*.V*. 

of  aggressins 

subcutane- 

ously. 

4 

Guinea-pig. 

6/IV  '05. 

i/ioo  loopful  of  swine  pest 

+3  c.cm.  sub- 

f    on     second 

subcutaneously. 

cutaneously. 

day. 

c 

Guinea-pig. 

6/rv  'CK. 

3  c.cm.  subcu- 

Remains alive. 

•J 

/  •*-  *         0 

taneously. 

6 

Guinea-pig. 

6/rv  '05. 

i/  1000    loopful    subcutane- 

+ 2  c.cm.  sub- 

On 7  /IV  very  ill; 

ously. 

cutaneously. 

on8/IVveryill; 

9/IV    very   ill; 

lo/IV    very 

thin;  u/IV  be- 

gins to  pick  up 

slowly  and  re- 

mains   alive. 

Marked  infil- 

tration  around 

point  of  injec- 

/ 

tion. 

36  ACTIVE   IMMUNIZATION. 

It  can  be  deduced  from  this  experiment  that  i/ioo  of  a  loopful  of  swine 
pest,  which  represents  i/io  of  a  fatal  dose  for  a  guinea-pig  by  subcu- 
taneous injection,  can  be  converted  into  an  acutely  fatal  dose  by  injecting 
the  aggressin  simultaneously  or  a  half  hour  before  the  experiment. 

The  aggressin  itself  is  only  slightly  toxic,  and  the  quantity  injected  is 
well  borne  by  the  guinea-pig.  Its  power  of  increasing  the  virulence  of  the 
infective  material  varies  directly  with  its  quantity,  i.e.,  the  greater  the  dose 
of  aggressin,  the  more  rapidly  is  death  occasioned.  If,  however,  only  small 
doses  of  the  culture  are  given,  and  in  addition  to  this,  the  aggressin  is  injected, 
the  animal  does  not  die,  but  becomes  exceedingly  ill,  thus  indicating  the 
effect  of  aggressins.  In  this  connection  it  might  be  well  to  add  that  the 
aggressin  may  be  given  twenty-four  hours  previous  to  the  time  of  infection. 

On  microscopical  examination  of  the  aggressin  exudate,  of  only  very  few 
cells,  but  a  great  number  of  bacteria  are  present.  The  bacteria  here  have 
increased  during  the  short  time  after  the  infection  to  a  far  greater  extent  than 
they  would  have  done  in  an  artificial  medium.  The  body,  continually 
combatting  against  their  increasing  toxicity,  finds  itself  powerless  when 
its  limited  fighting  capacity,  decreasing  in  proportion  to  the  rise  in  strength 
of  the  hostile  micro-organisms,  is  expended,  and  ultimately  succumbs  to  the 
infection.  During  the  struggle  between  the  protective  forces  of  the  organ- 
ism and  the  invading  bacteria,  many  of  the  latter  are  destroyed  and  these 
disintegrated  bacteria  are  found  within  the  exudate. .  From  this  fact  Wasser- 
mann  and  Citron  have  formed  the  conclusion  that  the  aggressins  are  not  as 
Bail  claimed,  secretory  products  of  live  bacteria  produced  during  the  con- 
flict between  the  bacteria  and  the  body  organism,  but  rather  the  products  of 
broken  down  bacteria.  Therefore,  Bail's  supposition  that  aggressins  are 
only  obtained  in  the  living  body  is  erroneous  and  can  be  shown  to  be  so  by 
the  fact  that  aggressins  may  be  reproduced  whenever  the  essential  require- 
ments can  be  had,  and  these  are : 

1.  Large  numbers  of  bacteria. 

2.  Non-poisonous  agents  which  can  disintegrate  these  bacteria. 
Aggressins  thus  obtained  are  known  according  to  Wassermann  and  Cit- 
ron, as  "artificial"  in  contrast  to  Bail's  "natural"  ones. 

Wassermann  and  Citron  Method  of  Obtaining  Artificial  Aggressins. 

Cultures  are  grown  in  mass  on  Kolle's  flask  plates.  A  Kolle's  agar 
plate  is  equivalent  to  twelve  agar  slants.  For  the  inoculation  of  these 
flasks  a  long  platinum  loop  is  needed  which  transfers  some  of  the  culture  to 
the  plate.  The  transferred  material  is  then  spread  over  the  entire  surface 
of  the  flask  by  a  large  triangular  platinum  loop.  The  latter  is  made  by  in- 
serting into  a  holder  both  ends  of  a  not  too  thin  platinum  wire,  about  20  cm. 
in  length  which  is  then  shaped  into  a  triangular  form.  While  still  red  hot, 


OBTAINING   ARTIFICIAL  AGGRESSINS.  37 

this  triangular  loop  should  be  introduced  into  the  flask  and  allowed  to  cool 
there.  Before  the  culture  is  spread,  it  is  advisable  to  bend  the  entire  loop 
to  a  slight  angle  by  pressing  it  against  the  upper  uncovered  wall  of  the  flask, 
thereby  preventing  the  hot  end  of  the  loop  holder  from  coming  in  contact 
with  the  agar  surface.  It  is  best  also  to  test  the  platinum  loop  upon  the 
surface  of  the  agar  in  order  to  ascertain  whether  it  is  still  too  hot. 

After  twenty-four  hours  of  incubation  there  is  usually  a  pronounced 
growth  upon  the  plates.  This  culture  is  then  washed  off  either  Fy  serum 
or  distilled  water  ("serous"  or  "aqueous  aggressin").  The  former  may 
be  obtained  fresh  from  a  rabbit.  Usually  10  to  12  c.c.  of  fluid  per  flask  is 
required;  3  or  /J.C.G.  are  first  poured  upon  the  culture  growth  and  the  mass 
scraped  gently  but  quickly  with  the  triangular  loop.  Then  the  remainder 
of  the  fluid  7  to  8  c.c.  is  poured  in  to  release  the  still  adherent  bacteria. 
The  turbid  milky  emulsion  is  collected  either  in  a  small  dark  glass  Erlen- 
meyer  flask  or  a  brown  bottle.  This  is  then  placed  into  a  proper  apparatus 
and  shaken  for  one  to  two  days  at  room  temperature.  Enough  carbolic 
acid  is  added  to  make  a  1/2  per  cent,  phenol  solution,  and  the  emulsion  is 
centrifugalized  and  sterilized  in  the  same  manner  as  has  been  described  for 
the  natural  aggressins. 

The  tendency  of  aggressins  towards  increasing  virulence  ("infektions 
beforderung  ")  is  the  same  whether  these  aggressins  are  artificial  or  natural. 

From  the  following  experiment  it  can  be  seen  that  the  bacteria  contain 
some  substance  which  is  easily  soluble  in  the  body  fluids  and  in  distilled 
water,  and  which  has  a  proclivity  toward  increasing  the  infectious  nature  of 
their  respective  bacteria  when  injected  simultaneously  with  them.  In  small 
doses,  this  substance  is  not  poisonous,  in  large  doses  it  may  be,  but  is  not 
necessarily  so.  There  is  no  definite  relation  between  the  poisonous  quali- 
ties of  the  aggressin  and  its  power  to  increase  the  virulence  of  an  infection. 
This  disproves  the  assumption  of  some  authors  that  the  action  of  the  ag- 
gressins is  dependent  upon  the  toxicity  of  the  endotoxins. 


ACTIVE    IMMUNIZATION. 

Experiment. 


No. 

Animal. 

Date. 

Amount  of  infec- 
tive material. 

Serous 
Aggressin. 

Watery 
Aggressin. 

Results. 

i 

Guinea-pig. 

2Q/V    '05. 

i/  200    loop     of 

Remains 

2 

Guinea-pig. 

20/V     'OS. 

swine  pest  sub- 
cutaneously. 
i/  200    loop     of 

+  2.5  c.c. 

alive. 
f  after 

7 

Guinea-pig. 

20/V     'CX. 

swine  pest  sub- 
cutaneously. 

subcutane- 
ously. 

+  2     SCC 

twenty-four 
hours 

4 

Guinea-pig. 

2/VI  '05. 

i/  200    loop     of 

subcutane- 
ously. 

2  c  c   subcu- 

alive, 
"i"  after  three 

c 

Guinea-pig. 

2/VI  '05. 

swine  pest  sub- 
cutaneously. 
i/  200    loop     of 

taneously. 
•?  c  c   subcu- 

days. 
"j~  after  three 

6 

Guinea-pig. 

2/VI  '05. 

swine  pest  sub- 
cutaneously. 

taneously. 
3  c  c   subcu- 

days. 
Remains 

7 

Guinea-pig. 

2/VI  '05 

taneously. 
A   cj  c  c  sub- 

alive. 

8 

Guinea-pig. 

2/VI  '05. 

i/  200    loop     of 

cutaneously. 

four  hours. 
Remains 

swine  pest. 

alive. 

Second  Fundamental  Aggressin  Test. 

(Its  Property  of  Active  Immunization.} 

Bail  and  his  pupils  believe  that  when  bacteria  invade  a  normal  organism, 
it  is  the  aggressin  power  of  these  bacteria  which  determines  whether  or  not, 
by  their  multiplication  disease  will  set  in.  If  it  does,  the  infection  continues 
until  the  "aggressive"  nature  of  the  bacteria  is  curbed.  As  there  are  some 
bacteria  which  on  injection  do  not  produce  any  disease,  Bail  attributes  this 
phenomenon  of  immunity  to  the  missing  "aggressive"  action  of  the  respective 
bacteria.  It  is  not  merely  the  presence  of  bacteria  which  is  the  criterion 
for  the  existence  of  disease;  as  long  as  they  are  void  of  their  "aggressive" 
property,  they  have  actually  become  saprophytes. 

Accordingly,  Bail  believes  that  the  bactericidal  immunity  is  no  true 
immunity  because  it  can  be  obtained  by  injection  of  dead  micro-organisms 
or  by  live  bacteria  in  such  minute  doses  that  no  specific  symptoms  are  pro- 
duced, i.e.,  no  aggressins  are  produced  within  the  body.  "If  the  immunity 
lacks  the  "anti-aggressive"  component,  which  alone  governs  the  existence  of 
disease,  one  gains  only  an  apparent  immunity  against  the  exciting  factor  of 
the  disease,  but  not  against  the  disease  itself." 


IMMUNITY  AGAINST   PURE    PARASITE.  39 

Bail  places  the  utmost  stress  upon  the  difference  between  an  immunity 
directed  against  the  exciting  agent  of  the  disease  (bactericidal  immunity)  and 
that  against  the  disease  itself  (anti-aggressive  immunity) . 

Immunization  against  the  disease  is  only  possible  if  the  aggressin  reaches  the  body 
of  the  animal  to  be  immunized.  This  is  possible  either  by  employing  Pasteur's  method 
of  vaccine  inoculation,  i.e.,  the  injection  of  bacteria,  the  "  aggressive"  nature  of  which 
has  been  weakened  but  not  destroyed,  or  by  direct  inoculations  of  aggressins.  The 
latter  is  by  far  the  simpler  and  more  reliable  mode  of  procedure,  being  productive  of 
a  true  immunity. 

Nowhere  does  this  problem  appear  of  such  extreme  importance  as  where 
immunity  against  a  pure  parasite  is  contemplated  as  in  the 
Immunity     case  °f  swine  pest  and  chicken  cholera.     While  it  is  exceed- 
Against  Pure  ingly  difficult,  in  fact  almost  impossible  to  immunize  against 
Parasite,      these  bacteria  either  with  dead  or  living  germs  or  vaccines, 
this  task  is  readily  accomplished  by  the  injection  of  non- 
poisonous  aggressins,  inasmuch  as  they  are  well  tolerated.     In  addition 
these  bacteria  are  of  help  in  definitely  deciding  whether  or  not  an  aggressin 
immunity  is  at  all  possible. 

Weil,  a  co-worker  of  Bail's,  has  carried  out  these  experiments  for  chicken 
cholera,  while  the  author  has  done  the  same  for  swine  pest. 


Example  of  Active  Immunization  with  Natural  Aggressins. 

a.  Slow  Immunization. 

Rabbit  I. 

6./IV.    1905  ist  injection:  i.o  c.cm.  natural  swine  pest  aggressin  intraperitoneally. 
17. /TV.  2d  injection:  i.o  c.cm.  natural  swine  pest  aggressin  intraperitoneally. 

25. /TV.  3d  injection:  i.o  c.cm.  natural  swine  pest  aggressin  intraperitoneally. 

i./V.  4th  injection:  2.0  c.cm.  natural  swine  pest  aggressin  subcutaneously. 

12. /V.  5th  injection:  2.0  c.cm.  natural  swine  pest  aggressin  subcutaneously. 

i6./VI.  ist  infection;  with  i/ioo  loopful  of  swine  pest  culture  intravenously. 

17. /VI.  Perfectly  well. 

8./VII.  2d  infection;  with  i  loopful  of  swine  pest  culture  intravenously. 

I5./VII.  Perfectly  well. 

22. /IX.  3d  infection;  with  i  loopful  of  swine  pest  culture  intravenously. 

3-/X.  Perfectly  well. 

Rabbit  I.  Controls.  Rabbit  II. 


i6./VL  1905  1/100,000  loopful  of  swine 
pest  culture  intra- 
venously. 

I7-/VI.  t  found  dead. 


8./VII.  1905  1/10,000  loopful  of  swine 
pest  culture  subcutan- 
eously. 

9./VII.  f  found  dead. 


40  ACTIVE    IMMUNIZATION. 

b.  Rapid  Immunization. 

Rabbit  II. 

8./IV.  1905  Injection  of  4  c.c.  of  natural  swine  pest  aggressin  subcutaneously. 
lo./IV.  Animal  somewhat  depressed. 

I3./IV.  Perfectly  active. 

26. /IV.  ist  infection:  i/io  loopful  of  swine  pest  culture  subcutaneously. 

i6./VI.  2d  infection:  i  loopful  of  swine  pest  culture  subcutaneously. 

Animal  remained  active. 

Controls. 

Rabbit  III. 

26. /TV.  1905  1/10,000  loopful  of  swine  pest  culture  subcutaneously. 
27./IV.  t- 

These  experiments  prove  conclusively  that  by  the  method  described  above, 
it  is  possible  to  attain  a  high  grade  of  immunity.  In  this  connection,  how- 
ever, it  is  very  important  to  adhere  to  what  Bail  pointed  out,  namely,  that 
a  long  period  should  elapse  between  the  last  inoculation  with  the  aggressin  and 
the  first  infection;  the  reason  for  that  being,  that  during  the  period  of  immuniza- 
tion, and  following  it  for  a  longer  duration  of  time,  there  is  a  condition  of 
hyper  susceptibility  to  infection. 

Example  of  Active  Immunization  with  Artificial  Aggressins. 

Rabbit  i. 

3./VI.    1905  ist  injection:  4  c.cm.  of  watery  extract  of  swine  pest  bacilli  subcutaneously. 
I4./VL  2d  injection:  2  c.cm.  of  watery  extract  of  swine  pest  bacilli  subcutaneously. 

25. /VI.  Removal  of  some  blood. 

4./VII.  3d  injection;  3  c.cm.  of  watery  extract  of  swine  pest  bacilli  subcutaneously. 

21  /VII.  Infection:  i/io  loopful  of  swine  pest  culture  subcutaneously. 

3-/X.  Animal  alive  and  healthy. 

Rabbit  2. 

19. /VI.    1905  ist  injection:  2 . 5  c.c.  of  serous  extract  of  swine  pest  bacilli  subcutaneously. 
9. /VII.  2d  injection:  2 .  o  c.c.  of  serous  extract  of  swine  pest  bacilli  subcutaneously. 

12. /VII.  3d  injection:  4.  o  c.c.  of  serous  extract  of  swine  pest  bacilli  subcutaneously. 

24. /VII.  ist  infection:  i/io  loopful  of  swine  pest  culture  subcutaneously. 

22./IX.  2d  infection:  i  loopful  of  swine  pest  culture  intravenously. 

Animal  remains  perfectly  well. 
Rabbit  3. 

i6./VI.    1905  Injection:  2.5  c.c.  of  watery  extract  of  swine  pest  bacilli  subcutaneously. 
4./VII.  Infection:  i/ioo  loopful  of  swine  pest  culture  subcutaneously. 

15. /VII.  Small  local  infiltrate.    Very  active. 

3-/X.  Animal  alive  and  perfectly  well. 

Control  animals  inoculated  on  the  days  of  infection  died  within  twenty-four  hours 
after  inoculations  of  1/100,000  loopful  of  culture  intravenously,  1/10,000  loopful 
subcutaneously. 

It  is  evident  from  the  above,  that  an  immunity  against  pure  parasites  can 
be  obtained  just  as  well  by  one  or  several  injections  of  extracts  of  living  bac- 


THIRD    FUNDAMENTAL  AGGRESSIN   EXPERIMENT.  41 

term,  as  by  injections  of  natural  aggressins.  As  the  possibility  of  the 
production  of  aggressins  by  a  struggle  between  the  bacteria  and  distilled 
water  can  be  excluded,  it  can  be  taken  without  further  explanation  that  in 
the  development  of  those  substances  which  have  a  tendency  to  increase  the 
virulence  of  bacteria,  or  which  can  be  used  to  produce  an  immunity,  the 
bacteria  play  a  passive  role,  in  that  they  are  only  extracted  by  the  dissolving 
agent.  The  difference  between  the  anti-bacterial  and  anti-aggressin  im- 
munity is  therefore  not  a  qualitative  one,  as  in  both  instances  it  is  the  sub- 
stances that  are  set  free  from  the  bacteria  which  stimulate  the  formation  of 
antibodies.  When  living  virulent  bacteria  are  injected  for  the  purposes  of 
immunization,  they  increase  so  rapidly  that  a  proper  dosage  is  impossible 
and  the  animals  frequently  die  before  enough  antibodies  are  liberated.  In 
addition  antibodies  are  also  generated  against  the  capsule  of  the  bacteria 
(bacteriolysins) . 

The  only  difference  between  immunization  with  morphologically  well 
preserved  but  dead  bacteria  and  that  with  aggressins  is  that  within  the  latter 
the  bacterial  substances  which  tend  to  bring  about  the  immunity  have  not 
been  altered  by  previous  heating,  but  exist  in  their  natural  easily  absorbable 
form.  Moreover,  by  using  the  extracts  one  does  away  with  certain  toxic 
substances  which  are  found  within  the  bacterial  capsules,  and  which  are 
rather  toxic  to  subcutaneous  tissue,  producing  necrosis  and  marasmus. 

The  Third  Fundamental  Aggressin  Experiment. 

Here,  it  is  demonstrated  that  the  serum  of  animals  immunized  by  aggres- 
sins either  artificial  or  natural,  contain  antibodies  which  (i)  can  neutralize 
that  property  of  aggressins  whereby  they  increase  the  virulence  of  bacteria; 
(2)  produce  a  passive  immunity  against  infection  with  living  bacteria. 

As  for  the  biological  structure  of  these  antibodies,  or  anti-aggressins  as 
they  may  be  called,  it  may  be  said  that  they  belong  to  the  class  of  ambo- 
ceptors,  shown  by  the  complement  fixation  methods. 

The  practical  employment  of  aggressins  as  a  method  of  immunization 
offers  distinct  advantages,  namely: 

1.  Absence  of  any  possible  dangerous  effects. 

2.  Absence  of  or  only  very  slight  local  and  general  reactions. 

3.  The  high  degree  and  long  duration  of  the  immunity  gained  by  pro- 
phylactic inoculations. 

4.  The  possibility  of  immunization  against  pure  parasites. 

5.  The  facility  with  which  the  inoculation  material  is  preserved. 
The  disadvantages,  however,  may  be  summarized  as  follows: 

1.  The  manufacture  of  the  inoculation  material  is  rather  complex  and 
with  some  pathogenic  bacteria  (pest),  not  without  danger. 

2.  The  increased  susceptibility  during  the  interval  between  the  inocu- 
lation and  the  onset  of  Immunity. 


42  ACTIVE    IMMUNIZATION. 

The  last  point  applies  not  only  to  aggressins,  but  equally  to  other  methods 
of  active  immunization.  In  times  of  an  epidemic,  aggressin  immunization 
should  never  be  undertaken. 

When  one  bears  in  mind  the  great  advantages  derived  from  the  employ- 
ment of  this  form  of  immunization,  its  extensive  use  should  be  expected; 
especially  so  as  animal  experimental  work  with  the  most  important  of  infec- 
tious bacteria:  typhoid,  cholera  (Bail),  colon  (Salus),  dysentery  (Kikuchi), 
staphylococcus  (Hoke),  has  proven  it  to  be  of  greater  or  less  success.  And 
it  is  therefore  no  false  prophecy,  to  say  that  this  method  will  be  employed 
more  and  more  frequently  in  the  future;  particularly  for  pest,  in  man,  results 
obtained  in  animal  experimentation  by  Hueppe  and  Kikuchi  have  more 
than  sanctioned  its  employment. 

Other  methods  of  immunization  based  upon  the  Aggressin  principles 

have  been  advocated,  but  none  have  attained  any  practical  significance. 

Mention  however  must,  in  passing,  be  made  of  the  work  of 

Brieger's      Brieger  and  his  co-workers  Mayer  and  Bassenge.     Brieger 

Bacterial     had  made  extracts  of  typhoid  and  cholera  bacilli,  in  the  main 

Extracts,      identical  with  artificial  aggressins.     As  far  as  his  sterilization 

was  concerned,  he  obtained  that  by  filtering  the  extract  through 

the  Pukal  filter.     One  should  remember  that  during  this  procedure  many 

important  substances  are  lost,  but  in  spite  of  this,  his  results  of  inoculation 

in  man  have  been  most  encouraging,  and  there  is  a  possibility  that  his  method 

may  take  the  place  of  Wright's  or  Pfeiffer  and  Kolle,  as  the  reactions  are 

very  much  milder. 

Entirely  different  from  the  extracts  of  living  bacteria  are  those  made 
from  previously  killed  ones.  Neisser  and  Shiga  among  others,  have  immu- 
nized against  half  parasites  in  this  manner.  This  is  not  surprising  as  the 
dead  bacter  al  bodies  can  be  similarly  used  for  this  purpose.  As  a  general 
rule,  wherever  dead  bacterial  bodies  cannot  be  used  for  immunization,  their 
extracts  will  also  be  found  inefficient.  The  oldest  bacterial  extracts  in  use 
are  the  tuberculins. 


CHAPTER  V. 
TUBERCULIN  DIAGNOSIS. 

As  a  member  of  the  class  of  bacterial  extracts,  tuberculin  merits  especial 
consideration,  because  it  is  used  not  only  for1  immunization,  but  also  for 
diagnostic  purposes.  Tuberculin  diagnosis  can  be  employed  in  three  ways. 

1.  As  Koch's  subcutaneous  method. 

2.  As  the  cutaneous  reaction  (v.  Pirquet)  and  ointment  reaction  (Moro). 

3.  As  ophthalmo  reaction  (Calmette) . 

Koch's  Subcutaneous  Method. 

In  the  chapter  on  aggressins  it  was  shown  that  when  a  normal  animal 
was  inoculated  with  a  certain  definite  quantity  of  bacterial  extract,  it  could 
readily  withstand  any  effects  of  such  inoculation.  If,  however,  a  similar 
quantity  was  injected  into  an  animal  previously  infected  with  the  same 
bacterium,  dangerous  symptoms  would  be  in  evidence  and  if  the  dose  were 
large  enough,  death  would  be  likely  to  follow. 

With  these  facts  for  reference,  the  following  experiments  will  be  easily 
understood.  A  number  of  tuberculous  guinea-pigs,  and  a  number  of  normal 
ones  as  control,  are  injected  with  varying  doses  of  tuberculin.  After  twenty- 
four  hours  some  of  the  tuberculous  animals  are  dead,  others  very  ill,  while 
the  normal  guinea-pigs  remain  perfectly  active.  Just  as  in  the  aggressin 
experiment,  we  have  here  a  bacterial  product  in  itself  possessing  only  slight 
toxic  qualities  which  has  so  increased  the  virulence  of  the  infection  already 
existing,  that  an  ailment  which  is  usually  of  a  slowly  progressive  nature 
becomes  transformed  into  an  acute  one,  terminating  in  the  death  of  the 
animal. 

The  close  analogy  between  the  experiments  with  aggressin  as  the  injected 
substance,  and  that  of  the  tuberculin,  will  become  more  clear  when  the  nature 
of  the  latter  is  perfectly  understood. 

Four  to  six  weeks  old  pure  cultures  of  the  tubercle  bacilli 

Derivation  of    grown  in  5  per  cent,  of  glycerin  bouillon  are  filtered,  and  the 

Tuberculin,     filtrate  then  evaporated  down  to  i/io  of  its  original  volume. 

The  resultant  fluid,  known  as  tuberculin,  is  dark  brown  and 

syrupy  in  nature,  and  has  the  quality  of  keeping  indefinitely. 

It  consists,  therefore,  as  we  see  of  a  50  per  cent,  glycerin  extract  of  the  soluble  products 
of  metabolism  of  the  tubercle  bacillus. 

43 


44  TUBERCULIN   DIAGNOSIS. 

A  part  of  the  glycerin  has,  however,  been  used  up  for  the  nutrition  of  the  bacteria  and 
thus  it  is  highly  probable  that  after  four  to  six  weeks  the  bouillon  contains  less  than 
5  per  cent,  glycerin  and  the  evaporated  solution  less  than  50  per  cent.  The  specific 
substances  contained  within  the  tuberculin  have  not  been  definitely  established.  As 
probable  elements,  however,  may  be  recorded,  products  of  secretion  of  the  living  bacteria, 
of  degeneration  of  the  dead  bacilli  and  finally  the  glycerin  soluble  substances  extracted 
from  the  bacterial  bodies  during  the  heating.  All  these  substances  no  doubt,  and  many 
others  about  which  we  lack  information,  are  directly  concerned  in  the  activity  of  the 
tuberculin. 

Among  the  many  unsolved  questions  which  here  present  themselves  may,  in  addi- 
tion be  mentioned  the  one,  to  the  effect:  whether  any  substances  exist  in  the  filtrate 
which  are  thermolabile,  and  therefore  destroyed  or  modified  by  the  heating?  Accord- 
ing to  Bail's  researches,  the  aggressin  of  the  tubercle  bacillus  differs  from  all  other 
aggressins  in  that  it  is  not  thermolabile  and  can  moreover  withstand  high  grades  of 
temperature.  In  spite  of  this  though,  attempts  to  eliminate  the  heating  during  the 
manufacturing  of  the  tuberculin  should  merit  consideration. 

If  merely  the  term  "Tuberculin,"  is  used  one  always  has  in  mind  the 
filtrate  tuberculin,  also  known  as  Old  Tuberculin. 

The  above  described  experiment  with  the  tuberculous  guinea-pigs  has 
its  analogy  in  the  use  of  tuberculin  in  the  case  of  man.  Here,  however,  in 
order  to  avoid  dangerous  symptoms  far  smaller  doses  of  tuberculin  are 
selected. 

If  therefore  of  two  individuals  one  is  tuberculous  and  the 

The  Tuber-   other  not,  and  both  are  injected  with  the  same  amount  of  old 

culin  Reaction  tuberculin  o.ooi  c.c.,  the  healthy  individual  remains  perfectly 

in  Man.      normal  while  the  tuberculous  person  shows  a  typical  symptom 

complex  which  can  be  described  under, 

1.  General  reaction. 

2.  Focal  reaction. 

3.  Local  reaction. 

The  General  Reaction  consists  of,  fever,  headache,  malaise,  nausea, 
insomnia,  cough  irritation,  palpitation,  etc.  The  most  constant  symptom 
is  increased  temperature;  the  other  manifestations  may  only  be  very  mild 
or  even  entirely  absent. 

The  Focal  Reaction  exhibits  evidences  of  a  fresh  inflammatory  process  in 
the  suspicious  or  old  tuberculous  foci.  In  cases  of  lupus,  laryngeal,  and  iris 
tuberculosis,  this  inflammatory  reaction  can  be  distinctly  seen.  In  pulmon- 
ary tuberculosis  the  previously  vague  physical  signs  may  now  become  defi- 
nite; rales  may  appear,  dulness  may  be  increased,  and  eventually  pains  in 
the  chest  may  arise. 

The  Local  Reaction  is  noticed  at  the  point  of  inoculation.  In  spite  of  the 
sterile  needle  and  thorough  disinfection,  the  skin  around  the  site  of  the 
injection  becomes  red,  swollen  and  painful.  That  this  is  not  due  to  dirt 
infection  is  proven  by  its  absence  in  non-tuberculous  individuals. 


DOSAGE    OF   TUBERCULIN.  45 

Of  the  three  types  of  reaction  the  general  and  focal  symptoms  are  the 
most  constant.  Both  are  so  characteristic  for  the  existence  of  tuberculosis, 
that  their  appearance  justifies  the  diagnosis.  In  practice,  however,  it  is  the 
general  reaction,  or  almost  exclusively  the  manifestation  of  fever,  which  is  taken 
as  the  guiding  symptom. 

The  focal  reaction  in  all  non-visible  tubercular  lesions  is  determined  by 
subjective  methods,  while  increase  in  temperature  is  alone  an  objective 
finding. 

In  carrying  out  the  tuberculin  test,  one  must  remember  several  practical 
points  which  are  of  help  for  the  correct  interpretation  of  the  results.  These 
may  be  summed  up  thus : 

Inasmuch  as  the  rise  of  temperature  is  of  diagnostic  importance,  no 
patient  with  any  fever  should  be  subjected  to  the  inoculation.  For  several  days 
previous  the  patient's  temperature  should  be  taken  every  three  hours  and 
only  if  the  temperature  does  not  exceed  37°  C.  per  axilla  should  the  tuber- 
culin diagnosis  be  undertaken. 

The  quantity  of  tuberculin  to  be  injected  is  also  of  the  utmost  consequence. 
Too  high  doses  should  be  avoided,  as  the  specificity  of  this 

Dosage  o      reactiOn,  like  all  other  biological  reactions,  is  limited  quanti- 
Tuberculm.          .  ....... 

tatively.     While  small  doses  of  tuberculin  will  give   a  rise 

of  temperature  only  in  tuberculous  individuals,  larger  doses  may  give 
the  same  rise  even  in  healthy  people.  In  addition,  too  large  doses  as  a  rule 
may  produce  a  general  reaction  which  might  be  very  severe  and  entirely 
injurious. 

The  dosage  advised  by  Robert  Koch  for  the  diagnostic  tuberculin  reaction  is 
as  follows: 

1.  o.oooi  c.cm.  T.  (for  very  weak  individuals  and  children). 

2.  o.ooi  c.cm.  T. 

3.  0.005  c.cm.  T. 

4.  o.oi  c.cm.  T. 

5.  o.oi  c.cm.  T. 

The  dose  chosen  at  the  first  injection  is  as  a  rule  i  mg.  T.  Very  weak 
individuals,  i.e.,  those  in  an  advanced  stage  of  tuberculosis  or  those  who  have 
experienced  a  recent  hemoptysis,  as  well  as  children  should  receive  an 
initial  dose  of  only  o.i  mg.  T.  Bandelier  and  Ropke  who  have  a  wide  experi- 
ence in  this  field,  advise  0.2  mg.  T.  as  the  primary  dose. 

Few  patients  show  a  distinctly  positive  fever  reaction  even  with  this 
small  dose;  by  a  positive  reaction  is  meant  an  increase  in  the  temperature 
so  that  the  latter  is  at  least  0.5°  C.  higher  than  the  highest  point  before  the 
injection.  If  the  temperature  has  not  increased,  the  reaction  is  negative, 
and  after  an  interval  of  two  to  three  days  of  normal  temperature  the  second 
inoculation  of  5  mg.  T.  is  given.  If  it  happens  occasionally  that  after  first 
inoculation  there  is  a  doubtful  reaction,  i.e.,  there  is  an  increase  of  0.2°  to 


TUBERCULIN   DIAGNOSIS. 


CHART  i . — Example  of  a  diag- 
nostic tuberculin  reaction. 


0.3°  C.  then  the  dosage  at  the  second  injection 
should  not  be  increased  to  5  mg.,  but  the  same 
amount  i  mg.  T.  is  to  be  repeated.  In  a  tuber- 
culous individual  this  repeated  injection  of  i 
mg.  frequently  results  in  a  distinctly  positive 
reaction,  while  in  a  non-tuberculous  patient  in- 
stead of  the  former  doubtful,  a  distinct  negative 
reaction  is  obtained. 

The  general  rules  given  for  the  first  inocula- 
tion also  apply  to  the  second  with  5  mg.  In  a 
doubtful  reaction  with  this  dose,  one  does  not 
directly  proceed  to  the  10  mg.  dosage,  but  the  5 
mg.  dose  is  repeated  and  only  after  a  negative 
reaction  with  the  repeated  5  mg.  dose  are  the 
10  mg.  injected  (see  accompanying  Chart  i). 
This  represents  the  maximum  amount  of  tuber- 
culin to  be  used  for  diagnostic  purposes.  Koch 
advises  repetit  on  of  this  dose  if  no  reaction  is 
obtained.  The  majority  of  authorities,  however, 
abstain  therefrom.  In  fact  some  investigators 
claim  that  a  reaction  obtained  after  inoculat  on 
of  10  mg.  cannot  be  considered  specific,  because 
there  is  a  class  of  non- tubercular  individuals 
that  responds  to  this  quantity  of  tuberculin. 

Most  tuberculous  persons  react  after  a  dose 
of  i  to  5  mg.  T.;  those,  however,  who  are  very  far 
advanced  or  who  suffer  from  severe  cachexia, 
remain  unresponsive  to  even  much  greater  doses ; 
in  addition,  patients  whose  serum  contains  anti- 
tuberculin,  do  not  react  because  the  inoculated 
tuberculin  is  quickly  neutralized. 

According  to  Loewenstein  the  tuberculin 

Loewenstein's    reaction  does  not  depend  so  much  upon 

Dosage        the  quantity  of  the  tuberculin,  as  upon 

Scheme.       the  frequency  that  it  is  injected.     He, 

therefore,  advises  that  the  same  amount, 
about  0.2  mg.  be  inoculated  four  times  during  the 
course  of  twelve  to  sixteen  days.  And  the  results  are 
that  in  by  far  the  greater  majority  of  tuberculous  patients 
a  typical  reaction  appears  after  the  third  or  fourth  in- 
jection. The  author  has  no  personal  experience  with 
this  method,  but  the  reports  of  other  authorities  do  not 
exhibit  as  favorable  results  as  those  claimed  by  Loewen- 
stein. 


CUTANEOUS    REACTION.  47 

As  for  the  technique  of  injection,  the  inoculation  is  always 

Technique  of    given  subcutaneously,  and  the  back  or  breast  is  the  best  site 

Tuberculin      for  it.     The  dilution  is  made  with  physiological  salt  solution 

Injection.       or  o>^  per  cent,  carbolic  solution,  and  it  is  advisable  to  make 

it  immediately  before  the  injection. 

In  interpreting  the  result  of  the  reaction  one  must  exclude 

Value  of      rises  of  temperature  due  to  extraneous  influences  such  as 

Reaction.     Angina,  Influenza,  etc.     Furthermore  there  are  individuals, 

especially  hysterical  ones,  in  whom  any  injection  as  such  is 

apt  to  produce  a  rise  of  temperature.     To  guard  against  such  a  possibility  an 

injection  of  physiological  salt  solution  should  be  made  and  thus  quiet  any 

suspicion  of  error. 

The  diagnostic  use  of  tuberculin  is  indicated  when  one  is 
Indications,    dealing  with  adults  who  present  clinical  symptoms,  or  clinically 
suspicious  symptoms  of  tuberculosis,  but  are  devoid    of    the 
presence  of  tubercle  bacilli  and  temperature. 

Tuberculin  is  contra  indicated  in  patients  with  high  fever, 
Contra-      and  during  or  shortly  after  hemoptosis  or  hematuria.     In 
indications,    epilepsy,  marked  cardiac  or  renal  affection,  arteriosclerosis, 
diabetes,  and  similar  conditions,  inoculation  should  be  under- 
taken only  under  the  strictest  indications  and  with  great  care. 

A  positive  general  reaction  means  that  the  individual  is  infected  with 
tuberculosis,  but  does  not  throw  any  light  upon  the  site,  the  extent,  or  the 
prognosis  of  the  infection.  The  focal  reaction  allows  the  diagnosis  of  the 
position  of  the  lesion. 

The  Cutaneous  Reaction. 

The  cutaneous  reaction  was  first  introduced  by  v.  Pirquet,  who  noticed 
that  by  scarification  of  the  skin  and  application  of  tuberculin,  tuberculous 
children  would  develop  a  distinct  papule  at  this  point,  while  in  non-tuber- 
culous conditions  such  a  reaction  would  be  absent. 

The  Technique  of  the  Cutaneous  Reaction. 

"The  patient's  forearm  on  the  inner  side  is  cleansed  with  ether;  two 
drops  of  the  pure  undiluted  old  tuberculin  are  placed  upon  the  skin  about 
10  cm.  apart,  and  then  the  skin  is  scarified  first  between  the  two  drops,  for 
the  purposes  of  a  control,  and  next  within  each  of  these  drops. — [A  boring 
scarifier  devised  for  this,  works  very  easily.]  Finally  a  piece  of  cotton  is 
placed  upon  each  of  these  drops  and  allowed  to  remain  there  for  ten  minutes, 
after  which  the  cotton  is  removed.  A  dressing  is  not  necessary." 

Interpretations  of  the  Reaction. 

Scarification  of  itself  produces  the  so-called  "traumatic  reaction''  i.e., 
a  small  wheel  with  a  rose  colored  margin  appears  around  each  of  the  three 


TUBERCULIN   DIAGNOSIS. 


points  of  scarification.     This  reaction  passes  away  after  several  hours  and 
only  a  small  scab  remains  surrounded  by  a  red  rim. 

This  "traumatic  reaction"  is  to  be  sharply  differentiated  from  the  "specific 
reaction"  The  latter  is  noticed  only  upon  the  upper  and  lower  points  where 
the  tuberculin  has  been  applied  and  consists  of  a  red,  indurated  papule  which 
rapidly  extends  in  size  and  elevation,  measuring  10  to  30  mm.  in  diameter. 
(Fig.  i,  Plate  I).  The  papule  may  be  round  or  have  irregular  margins. 
Scrofulous  children  show  small,  irregularly  raised  follicular  infiltrations 
around  the  specific  reaction.  This  is  known  as  the  "scrofulous  reaction" 
It  may  appear  as  early  as  within  three  hours,  but  usually  occurs  within 
twenty-four  hours.  It  arrives  at  its  maximum  within  forty-eight  hours; 
occasionally  it  is  delayed  and  may  fully  develop  until  the  third  or  fourth  day 
and  then  it  begins  to  fade.  Frequently  a  small  pigmented  spot  remains. 
General  and  focal  reactions  are  practically  absent. 

Moro's  Ointment  Reaction. 

Moro  and  Doganoff  found  that  a  50  per  cent,  ointment  of  tuberculin  in 
lanolin  rubbed  into  the  skin  without  scarification,  would  give  a  reaction 

which  consisted  of  small  nodular  or 
papular  efflorescences  after  the  na- 
ture of  Lichen  Scrophulosorum. 
Therefore,  in  accordance  with  the 
number  and  size  of  these  nodules 
as  well  as  the  time  of  their  appear- 
ance, three  grades  of  reaction  are 
described. 

In  carrying  out  the  reaction  the 
ointment  is  heated  to  25°  C.  and  a 
quantity  about  the  size  of  a  pea  is 
thoroughly  rubbed  into  the  skin  of 
the  abdomen  or  the  region  of  the 
mamilla,  for  almost  a  minute.  The 
diagnostic  value  of  the  reaction  is 
variously  interpreted. 

An  almost  analogous  reaction, 
described  independently  of  Moro, 
by  Lignieres  and  Berger  is  to  be 
found  in  thoroughly  rubbing  in  con- 
centrated old  tuberculin  into  the 
shaved  skin  of  tuberculous  cattle. 


FIG.  13. — Inoculation  with  tuberculin  for  the 
Pirquet  reaction. 


Historical. 


The  Ophthalmo  Reaction. 

At  the  discussion  which  followed  v.  Pirquet's  presentation  of  his  cuta- 
neous reaction,  Wolff-Eisner  remarked,  "that  by  instilling  some  10 
per  cent,  tuberculin  into  the  conjunctival  sac,  a  local  conjunctivitis  was 


TECHNIQUE    OF   REACTION.  49 

obtained  and  occasionally,  also  a  general  reaction.  The  marked  severity  of  the  reaction, 
however,  and  its  apparent  lack  of  specificity,  made  its  diagnostic  value  improbable." 
Calmette,  who  believed  that  Wolff- Eisner's  failure  in  obtaining  authentic  results  lay  in 
the  fact  that  glycerin  was  contained  in  the  old  tuberculin  employed  by  him,  went  to 
work  and  by  alcohol  precipitation  obtained  a  glycerin-free  dry  product,  which  he  used 
in  a  i  per  cent,  solution  equivalent  to  10  per  cent,  old  tuberculin.  It  was  he,  therefore, 
who  first  established  the  clinical  diagnostic  value  of  the  reaction.  But  his  hypothesis 
was  erroneous,  as  the  mild  reactions  which  he  obtained  were  not  due  to  the  absence  of 
glycerin,  but  because  the  Lille  tuberculin  made  use  of  is  much  weaker  than  trie  German 
preparation.  The  author  was  able  to  show  that  the  old  tuberculin  could  very  well  be 
used  for  the  Ophthalmo  reaction  if  instead  of  the  10,  a  i  per  cent,  dilution  was  made. 
Thus  employed,  the  reaction  is  exceedingly  mild  and  specific.  Eppenstein  later  advised 
a  2  and  4  per  cent,  dilution  in  cases  where  the  i  per  cent,  solution  gave  no  reaction. 

Technique  of  Reaction. 

It  is  of  extreme  importance  to  have  freshly  prepared  sterile  dilutions  of 
the  old  tuberculin  (Hochst  Farbwerke).  All  the  ready-for-use  preparations 
on  the  market  should  be  discarded.  This  applies  also  to  the  "  Tuberculin  Test" 
Calmette' *s  sold  by  Poulenc  Freres. 

The  mishaps  and  low  grade  of  specificity  often  ascribed  in  literature  to 
the  ophthalmo  reaction  can  in  a  greater  majority  of  cases  be  explained  by  the 
employment  of  preparations  other  than  the 
i  to  2  per  cent,  fresh  dilutions  of  the  old 
tuberculin  advocated  by  the  author,  and  in 
still  another  number  of  cases  to  its  employ- 
ment in  conditions  where  it  was  distinctly 
contraindicated.  The  preparation  of  fresh 
tuberculin  dilutions  is  very  much  simplified 
by  the  "  Qphthalmodiagnosticum  for  Tuber- 
culosis" of  the  firm  P.  Altmann,  Berlin 
N.  W.  6  (Fig.  14). 

This  outfit  consists  of  twelve  sealed  glass 
tubes  each  containing  o.i  c.c.  old  tuberculin; 
a  cylinder  for  the  dilution  graduated  in  per-  FlG-  I4-~ Ophthalmodiagnosticum  for 

tuberculosis.     (After  Citron.') 

centages,  and  a  measuring  pipette  o.i  c.c. 

in  size  fitted  with  a  rubber  bulb.  One  of  the  sealed  ampoules  is  shaken 
so  that  the  tuberculin  is  collected  into  its  broader  part  and  then  broken  at 
the  designated  point  near  the  narrow  end.  The  tuberculin  is  drawn  up  to 
the  mark  into  the  pipette  and  then  transferred  into  the  cylinder.  Boiled 
water  or  sterile  saline  is  added  to  the  i,  2  or  4  per  cent,  dilution  mark. 
The  pipette  is  washed  clean  in  the  solution  by  successive  aspiration  and 
expulsion  in  order  to  free  it  completely  of  the  remaining  concentrated 
tuberculin,  and  can  now  be  employed  as  the  eye  dropper. 

The  solution  should  be  used  only  on  the  day  it  is  prepared.     The  tuber- 

' 


50  TUBERCULIN   DIAGNOSIS. 

culin  in  the  sealed  tube  can  be  kept  indefinitely.     The  pipette  and  graduate 
are  sterilized  by  dry  heat,  boiling  or  by  thorough  washing  in  boiling  water. 

One  drop  of  the  tuberculin  dilution  is  deposited  into  the  inner  angle  of 
the  eye,  and  care  should  be  taken  that  the  drop  is  not  immediately  expelled, 
but  evenly  distributed  in  the  conjunctival  sac. 

In  tuberculous  individuals  the  reaction  appears  in  twelve  to 
Gradation  of  twenty-four   hours,   and   according   to   its   intensity   can   be 
the  Ophthal-  divided  into  three  grades, 
mo  Reaction.  pirst  Grade. — Reddening  of  the  caruncle  and  inner  side   of 

the  lower  lid  (+)  (see  Fig.  2,  Plate  I).  . 

Second  Grade. — Same  as  above  but  additional  involvement  of  the  con- 
junctiva of  the  eyeball  (-f  +). 

Third  Grade. — Conjunctivitis  purulenta,  phlyctenulae  and  other  such 
severe  manifestations  (  +  +  +). 

The  reactions  of  the  first  and  second  degree  occur  most  frequently.     The 
manifestations  associated  with  the  former  of  these  are  so  mild  that  the  pa- 
tient himself  does  not  usually  notice  them.     If  the  proper  dilution  is  used  and 
the  contraindications  of  this  test  are  observed,  a  reaction  of  the  third  degree  is 
obtained  only  in  exceptional  cases.   Fever  never  occurs.   The  other  eye  serves 
as  a  control.     It  is  advisable  therefore  before  undertaking  the  reaction,  to 
note  carefully  any  differences  that  may  exist  in  the  conjunctivas  on  both  sides. 
It  must  be  remembered  that  i.  The  greater  the  dilution,  the 
Selection      more  specific  is  the  reaction. 

of  Correct     2.  The  test  should  not  be  repeated  upon  the  same  eye,  even 
Dilution,      if  there  was  no  reaction  at  all  at  the  first  instillation. 

The  following  procedure  should  be  adopted.  A  drop  of  the 
2  per  cent,  tuberculin  dilution  is  placed  within  the  left  eye.  If  a  positive 
reaction  takes  place,  it  is  of  great  probability  that  the  patient  is  suffering  from 
an  active  tuberculous  process  and  thus  the  diagnosis  is  established.  If, 
however,  that  proves  insufficient,  and  further  corroboration  is  required, 
the  patient  should  receive  after  the  first  reaction  has  entirely  subsided  one 
drop  of  a  i  per  cent,  tuberculin  dilution  into  the  right  eye. 

If  a  negative  reaction  is  obtained  at  the  instillation  of  the  2  per  cent,  dilu- 
tion, one  drop  of  the  4  per  cent,  dilution  is  placed  into  the  right  eye.  A 
negative  reaction  with  the  4  per  cent,  mixture  speaks  almost  conclusively 
for  the  absence  of  tuberculosis  except  in  far  advanced  cachectic  condi- 
tions. A  positive  result  does  not,  on  the  other  hand,  indicate  the  presence 
of  tuberculosis,  as  there  are  many  normal  individuals  who  react  to  a  4  per 
cent,  tuberculin  concentration. 

The  ophthalmo  reaction  is  indicated  in  all  suspicious  cases  of  tuber- 
Indications    culosis  where  the  presence  of  bacilli  cannot  be  demonstrated 
for  Ophthal-  anc[  wnere  the  subcutaneous  reaction  either  on  account  of  the 
eactions.  presence  of  temperature  or  other  reasons  cannot  be  undertaken. 


SPECIFICITY   OF    TUBERCULIN   REACTION.  51 

This  test  is  much  milder  and  more  agreeable  to  the  patient  than  the  sub- 
cutaneous one,  and  in  ambulatory  work  more  significant,  inasmuch  as  it  does 
away  with  any  necessity  for  considering  as  a  guide  the  temperature  taken 
by  the  untrained  and  usually  unreliable  patient. 

The  ophihalmo  reaction  is  contraindicated  in  all   diseases   of 

Contraindi-    ^6  eye,  tuberculous  or  otherwise.     If  one  eye  only  is  affected, 

cations  for  the  the  reaction  should  not  be  undertaken  upon  the  healthy  eye. 

Ophthalmo    Similarly,  patients  who  have  had  some  eye  disease,  even  though 

Reaction.     many  years  ago,  those  who  by  reason  of  their  occupation  are 

readily  exposed  to  eye  diseases,  or  who  live  in  districts  where 

trachoma  is  prevalent  should  be  excluded  from  the  test.     The  reason  being 

that  in  those  individuals  the  conjunctival  mucous  membrane  becomes  a 

locus  minoris  resistentiae  and  therefore  easily  inflamed 

Repeated  instillations  of  tuberculin  into  the  same  eye,  may  set  up  very 
severe  disturbances.  Scrofulous  children  often  show  reactions  of  the  third 
degree,  inasmuch  as  they  possess  the  constitutional  tendency  which  makes 
them  easily  susceptible  to  conjunctivitis  or  phlyctenulae.  In  patients  with 
a  positive  ophthalmo  reaction  which  has  subsided,  a  recurrence  of  the  con- 
junctival inflammation  is  frequently  observed  when  they  begin  to  receive 
subcutaneous  inoculations  of  tuberculin  for  therapeutic  or  even  diagnostic 
purposes. 

The  Specificity  of  the  Tuberculin  Reaction. 

The  one  real  essential  for  the  practical  application  of  all  biological  reactions,  is 
the  specificity  of  the  same.  There  is,  however,  as  will  be  repeatedly  pointed  out 
further  on,  no  single  absolutely  specific  reaction.  In  fact,  it  would  be  more  exact  to  con- 
sider these  biological  reactions  only  relatively  specific;  the  latter  depending  upon  the 
quantity  of  the  required  antigen  and  the  reacting  organism.  In  this  connection  it 
may  also  be  said,  that  it  is  never  possible  to  draw  an  exact  line  between  the  specific  and 
non-specific  biological  reactions.  There  always  will  be  a  doubtful  zone.  As  a  general 
rule,  however,  it  may  be  said  that  the  smaller  the  quantity  of  antigen  that  is  required 
and  the  stronger  the  resulting  reaction,  the  more  probable  is  the  biological  specificity. 

In  tuberculosis  this  problem  is  rendered  still  more  complex  by  the 
pathological  anatomical  findings,  whereby  it  is  shown  that  an  extraordinary 
high  percentage  of  individuals  have  undergone  tubercular  infection  at  some 
time  during  life.  The  clinical  consideration  of  tuberculosis,  however,  does 
not  deal  with  the  diagnosis  of  these  harmless,  practically  healed  tuberculous 
foci;  what  the  clinician  desires  to  know  is  whether  or  not  a  group  of  symptoms 
manifested  in  a  patient  is  of  a  tuberculous  nature  or  not.  In  other  words, 
it  is  not  the  latent,  inactive,  but  the  active  form  of  tuberculosis  that  is  to  be 
diagnosed.  If,  therefore,  one  views  the  various  tuberculin  tests  from  such  a 
stand-point  as  this,  he  arrives  at  very  material  differences. 

The  reaction  of  least  specificity  in  adults  is  the  v.  Pirquefs  cutaneous 
reaction.  In  children  it  is  far  more  specific. 


52  TUBERCULIN   DIAGNOSIS. 

Apropos  this  latter,  v.  Pirquet  makes  some  very  interesting  observations. 
Out  of  747  children  in  Escherich's  clinic  in  Vienna  upon  whom  the  reac- 
tion was  tried,  there  were: 

clinically  tuberculous  130,  out  of  which  113  (87%)  showed  a  positive  reaction; 

clinically  non-tuberculous  512,     "    "        "      104  (20%)        "       "        " 
doubtful  115,     "    "       "        56(48.6%)     "       "        " 

Almost  all  of  the  tuberculous  children  who  did  not  react  were  cachectic. 

As  for  the  positive  reaction  in  non-tuberculous  cases,  the  age  of  the  child 
in  large  part  explains  the  great  differences  found. 

Whereas  healthy  infants  up  to  the  sixth  month  almost  never  give  a  posi- 
tive reaction,  healthy  children  of 

1  to  2    years  react  in  2    per  cent,  of  cases. 

2  to  4    years  react  in  13  per  cent,  of  cases. 
4  to  6    years  react  in  17  per  cent,  of  cases. 
6  to  10  years  react  in  35  per  cent,  of  cases. 
10  to  14  years  react  in  55  per  cent,  of  cases. 

In  adults  one  meets  with  a  positive  v.  Pirquet's  reaction  in  more  than 
70  per  cent,  of  all  cases.  V.  Pirquet  explains  this  by  the  presence  of  latent 
tuberculosis. 

//  therefore  becomes  self  evident,  that  the  cutaneous  reaction  in  adults  is  void 
of  any  diagnostic  value.  A  negative  reaction  only,  can  be  fully  relied  on, 
and  that,  if  no  cachexia  exists. 

In  young  children  on  the  other  hand,  v.  Pirquet's  method  should  be  the 
choice.  In  addition  to  its  being  entirely  harmless,  and  easily  applied,  it 
possesses  a  high  diagnostic  value. 

As  for  Koch's  subcutaneous  reaction,  it  is  specific,  inasmuch  as  it  is  a  rare 
exception  to  get  a  negative  reaction  in  an  active  tuberculous  process.  This 
occurs  only  in  cases  either  with  very  severe  cachexia  or  those  with  freely 
circulating  anti-tuberculin  in  the  blood.  If  the  latter  two  possibilities  are 
excluded,  the  absence  of  a  positive  reaction  speaks  decidedly  in  favor  of  the 
absence  of  tuberculosis. 

The  interpretation  of  a  positive  reaction  as  to  the  existence  of  clinically 
active  tuberculosis  cannot  be  so  definitely  answered.  From  the  work  of 
most  of  recent  authorities,  however,  it  seems  to  be  taken  for  granted  that  a 
positive  reaction  does  mean  an  active  tuberculosis;  still,  this  statement 
requires  a  great  deal  of  consideration  and  limitation  as  well. 

In  this  connection  the  statistics  of  Franz  are  of  great  interest.  Out  of 
400  apparently  healthy  soldiers  in  one  of  the  Austrian  regiments  who  in  1901 
— their  first  year  of  service,  received  an  inoculation  of  0.003  c-c-  °f  tuberculin, 
a  positive  result  was  found  in  61  per  cent,  of  the  cases.  In  the  following 
year  (1902)  100  of  the  soldiers  were  re-inoculated  and  all  of  those  who  re- 
acted positively  the  first  time,  did  so  a  second  time,  in  some  instances  even 
though  the  second  dosage  was  smaller.  Moreover,  fourteen  others  who 


HEALTH   OF   INOCULATED    SOLDIERS. 


53 


responded  negatively  the  previous  year  showed  positive  results  this  time, 
making  a  total  of  76  per  cent.  Out  of  323  men  inoculated  for  the  first  time 
in  1902,  68  per  cent,  reacted  positively.  It  must  be  mentioned,  however, 
that  the  majority  of  members  of  this  regiment  came  from  a  very  tuberculous 
district.  The  same  author  also  examined  a  Hungarian  regiment  in  a 
tuberculous-free  district,  and  under  similar  circumstances  found  a  positive 
reaction  in  38  per  cent,  of  cases.  Although  these  figures  may  be  excep- 
tionally high,  they  are  without  doubt  conclusive  as  to  the  fact  that  Koch's 
reaction  in  this  respect,  cannot  be  considered  specific  for  "active"  tubercu- 
losis. Franz  in  addition  gives  important  statistics  concerning  the  health  of 
the  inoculated  soldiers  whom  he  examined  for  years  following  the  inocula- 
tion. The  following  charts  taken  from  the  most  recent  publication  of  Franz 
(Wien.  Klin.  Woch.,  1909,  No.  28)  tabulates  what  has  been  said  above. 


!  During  the  period  of   three  years,    those 

Regiment. 

Year  of 
injection 

No.  of 
soldiers 
inocu- 

Positive reaction. 

that  terminated  their    service    through 
death,    invalidity   or    longer    leave   of 
absence  showed. 

lated. 

Negative  reaction. 

Tuberculosis. 

Disease  suspi- 
cious of  tub. 

Other 
diseases. 

Bosn.  Inf. 

1901 

400 

/  +245  (61%) 

17  (8  deaths) 

22 

10 

Reg.  No.  i. 

\  -155(39%) 

5  (4  deaths) 

25 

7d) 

Bosn.  Inf. 

1902 

323 

/    +222   (68.7%) 

13  (6  deaths) 

28(1) 

7(2) 

Reg.  No.  i. 

\  -ioi  (31.3%) 

4 

*3 

5 

Inf.    Reg. 

1902 

279 

/    +I08(38.7) 

4 

4 

8 

No.  60. 

\    -171   (61.3) 

3  (2  deaths) 

5 

12 

Total  

IOO2 

(  +C7* 

34  (14  deaths) 

54(i) 

25  (2) 

\  -427 

12  (  6  deaths) 

43 

24(l) 

Those  who  reacted  in  1901 

Regiment. 

Time  of 
observation. 

No.  ill  with 
manifest 
tuberculosis. 

and  1902  to  3  mg.  tuber- 
culin. 

Positive. 

Negative. 

Bosn.  Inf.  Reg.  No.  i.     I  Ser. 

From    10.    x.    '04 

10 

6 

4 

(400  men). 

until  end  of  1908. 

Same.     II  Ser.  (323  men);  Inf. 

From    Oct.,    1908, 

6 

5 

i 

Reg.  No.  60  (279  men). 

until  end  of  1908. 

2 

j 

i 

18 

12 

6 

54 


TUBERCULIN   DIAGNOSIS. 


In  regard  to  the  specificity  of  the  ophthalmo  reaction,  the  condi- 

,  tions  here  are  more  favorable  than  in  both  of  the  preceding 

of  Ophthalmo  A  , 
Reaction      tuberculin  tests.     The  following  short  chart  is  explanatory. 

Positive  reactions  were  obtained  in 


Aude"oud. 

Petit. 

Citron 
(ist  series). 

Eppenstein. 

Schenk  and 
Seiffert. 

Tuberculosis    . 

O4   6% 

QA     7% 

80  7% 

72    1°7^ 

78  6% 

Suspicious  cases.  . 
Normal  cases.  .  .  . 

81.0% 
8-3% 

61.6% 
18.4% 

ou  •  /  /o 
80.0% 

2-2% 

.     1  *  •  3  /O 

40.0% 
9-o% 

/  o  .  u  /Q 
30-0% 
5-8% 

Calmette's  preparation, 


i%  old  tuberculin. 


It  is  evident  from  the  above  figures  that  by  the  use  of  the  i  per  cent,  tuber- 
culin a  grade  of  specificity  is  reached  which  can  be  considered  quite  high,  as 
the  non-tuberculous  react  only  in  a  very  small  percentage  of  cases,  while  existing 
tuberculosis  is  detected  in  80  per  cent,  of  the  subjects.  Clinical  examinations 
of  the  positive  reacting  patients  show  that  the  latter  belong  to  the  group  of 
active  tuberculosis.  Absolute  reliance,  however,  in  the  determination  as 
to  whether  the  positive  reaction  given  is  due  to  an  active  or  latent  tuberculosis, 
cannot  even  be  placed  upon  the  ophthalmo  reaction. 

According  to  several  authors,  it  is  claimed  that  typhoid  fever,  rheumatism,  and 
syphilis  (in  the  stage  of  eruption)  are  very  prone  to  give  a  positive  ophthalmo  reaction, 
without  the  presence  of  a  simultaneously  existing  tuberculosis. 

In  conclusion,  therefore,  the  author  finds  it  difficult  to  make  any  general 
statement  as  to  the  preference  of  one  or  the  other  reaction  test  for  diagnostic 
purposes. 

In  children,  however,  it  may  be  said  that  the  application  of  the  Pirquet 
reaction,  in  adults,  the  ophthalmo  reaction,  are  given  preference  to  Koch's 
reaction,  provided  no  contraindications  exist  against  the  former,  and  that 
treatment  with  tuberculin  is  not  to  be  undertaken.  In  the  latter  instance, 
the  recurrent  ophthalmo  reaction  when  the  tuberculin  therapy  is  instituted, 
authorizes  the  use  of  Koch's  subcutaneous  diagnostic  method. 

Mallein,  Trichophytin. 

Similar  to  old  tuberculin,  the  Mallein  (Helmann  and  Kelning)  has  been  obtained 
from  cultures  of  Glanders  bacilli  and  the  Trichophytin  (Plato)  has  been  isolated  from 
the  Trichophyton  fungi.  Mallein  has  already  attained  a  place  in  practical  application 
for  the  diagnosis  of  glanders  in  veterinary  medicine.  Like  tuberculin  it  is  harmless  in 
normal  organisms,  but  brings  about  temperature  and  a  local  reaction  at  the  site  of  the 
injection  when  inoculated  into  glanders  stricken  animals.  Various  general  symptoms 
may  also  appear.  Its  employment  in  a  manner  analogous  to  the  ophthalmo  reaction 
is  also  possible. 


CHAPTER  VI. 
THE  TUBERCULIN  THERAPY. 

Right  at  the  beginning  it  must  be  made  clear,  that  the  use  of  tuberculin 
is  not  to  be  considered  as  a  curative  agent  against  tuberculosis,  but  rather 
in  the  light  of  a  bacterial  extract  for  active  immunization.  In  the  previous 
chapter  it  has  been  shown  that  while  there  are  some  infectious  diseases 
where  immunization  can  be  accomplished  by  the  use  of  bacterial  extracts 
and  dead  bacteria,  there  are  others  where  immunization  is  possible  only 
when  living  vaccines  or  aggressins  of  living  bacteria  are  employed.  In 
both  of  these  instances,  however,  healthy  individuals  are  being  treated  to 
be  protected  from  future  infection.  An  exception  is  presented  by  rabies. 
In  this  disease,  the  vaccination  against  the  active  symptoms  is  instituted 
after  the  infection  has  already  taken  place,  but  the  redeeming  feature  about 
its  treatment  is  the  existence  of  the  very  long  incubation  period.  Thera- 
peutic use  of  tuberculin,  however,  is  a  form  of  active  immunization  which 
belongs  to  neither  of  the  above  classes.  The  principle  involved  here  is 
entirely  different,  and  the  question  arises  if  it  is  at  all  possible  to  obtain  an 
active  immunity  by  the  injection  of  antigen  in  a  condition  where  infection 
has  already  taken  place,  and  produced  pathological  changes.  [In  other 
words,  where  spontaneous  immunization  has  failed.] 

An  answer  to  this  question  is  to  be  found  in  Koch's  fundamental  experi- 
ments which  have  been  the  basis  as  well  as  starting  point  of  the  entire  tuber- 
culin study. 

If  a  normal  guinea-pig  is  inoculated  with  tubercle  bacilli,  the  point  of  inoculation 
very  soon  closes.  After  ten  to  fourteen  days  there  appears  at  this  site  a  small  hard 
nodule  which  finally  ulcerates.  This  shows  no  tendency  to  heal  and  remains  so  until 
the  death  of  the  animal.  If,  however,  an  already  tuberculous  guinea-pig  is  similarly 
inoculated,  while  the  point  of  inoculation  also  closes,  no  indurated  nodule  appears. 
Instead,  a  necrotic  process  of  the  skin  sets  in  after  the  second  day,  which  finally  terminates 
in  the  casting  off  of  the  slough  and  the  formation  of  a  flat  ulceration  that  heals  rapidly. 
It  does  not  matter  at  all  whether  living  or  dead  tubercle  bacilli  are  used  for  the  second 
infection. 

In  explanation  of  the  above  phenomenon  it  must  be  said  that  the  first 
injection  although  it  had  a  fatal  effect  upon  the  animal  must  have  stimulated 
certain  immune  reactions  within  the  organism  which  became  manifest 
after  the  second  inoculation.  That  a  condition  similar  to  this,  or  even 
more  favorable  exists  in  man,  is  proven  by  the  fact  that  while  the  large  majority 

55 


56  THE  TUBERCULIN  THERAPY. 

of  people  become  infected  with  tuberculosis  at  some  time  during  their  lives, 
only  a  small  proportion  show  symptoms  referable  to  the  disease  and  the 
other  greater  number  undergo  spontaneous  cure. 

Koch  further  showed  that  the  injection  of  tuberculous  guinea-pigs  with 
large  doses  of  tubercle  bacilli  produced  rapid  death,  while  frequently  repeated 
small  doses,  evinced  favorable  effects  upon  the  site  of  injection  and  the  general 
condition  of  the  animals.  In  this  way  he  proved  the  beneficial  influence 
which  successive  inoculations  exert  upon  the  primary  infection. 

In  the  employment,  however,  of  dead  tubercle  bacilli  in  man  for  the  purpose 
of  therapeutic  injections,  a  serious  difficulty  presented  itself.  It  was  found 
that  the  inoculated  dead  bacilli  were  not  absorbed,  but  remained  for  a  long 
time  at  the  seat  of  the  inoculation  instigating  suppurative  processes.  On 
intravenous  application,  formation  of  tubercular  nodules  was  noticed. 

Koch  realized  that  these  harmful  effects  were  due  to  the  non-absorbable 
parts  of  the  tuberc'e  bacilli;  in  the  main  the  bacterial  capsules.  He  therefore 
attempted  to  extract  the  immunizing  substances,  and  in  this  way  brought 
about  the  existence  of  old  tuberculin. 

Questions  may  here  be  asked  to  the  effect,  whether  this  old  tuberculin  is  identical 
with  tuberculous  antigen;  whether  it  is  at  all  a  feasible  preparation  for  purposes  of 
immunity;  does  it  contain  all  the  important  elements  of  the  tubercle  bacillus,  if  not 
which  are  lacking?  The  specificity  of  immunity  reactions  has  already  been  dwelt  upon 
sufficiently  to  make  it  clear  that  immunizing  a  healthy  individual  with  old  tuberculin 
will  bring  about  an  immunity  only  against  the  substances  contained  within  this  prepa- 
ration. That  that  does  not  meet  the  requirement  is  proven  by  the  fact  that  an  animal 
immunized  against  tuberculin  will  not  be  protected  against  a  later  infection  with  living 
tubercle  bacilli.  It  cannot  therefore,  be  expected  that  immunization  of  a  tuberculous 
individual  with  old  tuberculin  will  protect  him  against  living  tubercle  bacilli.  The 
expectation,  however,  that  his  immunity  will  be  raised  against  old  tuberculin  only,  is 
fully  borne  out. 

Furthermore,  we  have  seen  that  in  the  aggressin  experiments,  inoculation  of  animals 
with  the  aggressin  antigen  was  sufficient  to  increase  the  immunity  so  that  a  subsequent 
infection  was  not  attended  by  any  harmful  effects.  In  this  case  the  injected  living  bacteria 
are  not  destroyed,  but  their  ill  effects  upon  the  immunized  organism  have  been  paralyzed. 
In  other  words,  the  parasites  have  been  transformed  to  saprophytes.  That  a  similar 
state  of  affairs  exists  in  the  use  of  antitoxic  sera  will  readily  be  seen.  The  antitoxic 
diphtheria  serum,  for  example,  neutralizes  the  toxin  and  thus  cures  the  disease.  The 
bacteria  themselves,  however,  remain  intact  and  also  infectious  for  untreated  individuals. 
Only  later  on  are  they  absorbed  by  the  phagocytes.  When  therefore  in  an  individual 
who  has  passed  through  a  course  of  tuberculin  treatment  there  are  found  fully  virulent 
tubercle  bacilli  in  the  sputum,  it  is  no  proof,  if  that  is  the  only  corroborative  evidence, 
that  the  tuberculin  treatment  had  been  inefficient.  In  fact,  there  are  strong  possibilities 
that  the  tubercle  bacilli  have  become  transformed  into  saprophytic  bacteria.  It  is,  how- 
ever, a  noteworthy  and  important  fact,  that  immunization  with  tuberculin  proves  no 
protection  against  later  infection  with  living  tubercle  bacilli,  while  in  the  case  of  aggressins 
and  toxins  this  is  possible. 

Although  tuberculin  cannot  be  considered  as  the  aggressin  or  toxin  of  the 
tubercle  bacilli,  it  simulates  these  substances  with  sufficient  closeness  to 


TUBERCULIN   PREPARATIONS.  57 

warrant  its  use  in  tuberculosis.  It  brings  about  an  immunity  against  some 
of  the  poisonous  products  of  the  tubercle  bacillus,  leaving  the  others  to  be 
combatted  by  the  natural  fighting  powers  of  the  individual. 

The  knowledge  that  this  old  tuberculin  represents  only  a  partial  aggressin, 
Various       or  toxin,  and  by  that  is  meant  that  it  does  not  contain  all  the  necessary 
Tuberculin     elements  for  the  establishment  of  a  true  immunity,  has  led  to  the  pro- 
Preparations,  duction  of  a  large  group  of  preparations  which  are  supposed  to  supply 

the  missing  properties  of  the  old  tuberculin. 

The  most  important  of  these  preparations  was  made  by  Robert  Koch.  Those  which 
are  of  frequent  use  are: 

a.  Old  tuberculin  (T.  Tuberculin) — preparation  described  on  page  47. 

b.  Original  old  tuberculin  (T.  O.  A.  Tuberculin  Original  Alt.) 

The  latter  consists  of  the  original  nitrate  of  the  tubercle  bouillon  culture  and  varies 
from  the  old  tuberculin  in  that  it  is  not  heated  and  reduced  to  i/io  its  volume.  The 
omission  of  heating  is  certainly  not  without  effect,  inasmuch  as  high  heat  without 
doubt  modifies  in  some  way  the  soluble  bacterial  substances.  This  preparation  has 
not  been  used  therapeutically  by  Koch  himself.  Spengler  and  especially  Denys,  who 
have  made  wide  use  of  it  under  the  name  of  "Le  bouillon  nitre,"  have  been  its  main 
supporters. 

c.  Vacuum  tuberculin  (V.  T.)  is  the  original  tuberculin  which  has  been  reduced  in 
vacuum  to  i/io  its  volume. 

d.  The  aqueous  tuberculin  Maraglianos  (Tuberculina  Aquosa)  is  closely  allied  to 
the  above  tuberculins.     It  contains  all  the  water  soluble  extracts  of  the  living  tubercle 
bacilli  obtained  by  extraction  of  the  living  bacteria  in  distilled  water,  followed  by 
filtration.     As  is  evident,  it  is  prepared  on  the  same  principle  as  Brieger's  bacterial 
extracts  and  Wassermann- Citron's  artificial  aggressins. 

The  above  mentioned  tuberculin  preparations  are  all  very  much  alike 
in  that  they  contain  the  soluble  bacterial  elements.  Their  action  therefore 
corresponds  more  or  less  to  that  of  old  tuberculin. 

Another  set  of  preparations  have  as  their  basis  the  insoluble 

New         bacterial  substance  which  cannot  as  such,  in  either  living  or 

Tuberculin    dead    form,    be    absorbed.     Since,   however,  the  absorption 

Preparations.  of  bacteria  is  a  prerequisite  to  their  proper  action,  it  was 

necessary  to  so  alter  the  body  substances  of  these  bacteria  that 

they  could  be  taken  up.     Koch  found  that  this  was  best  accomplished  by 

thoroughly  pulverizing  the  bacilli  in  large  mortars.     And  by  this  means  the 

first  preparation  which  he  obtained  was 

e.  New  tuberculin  T.  R.   (Koch)  Tuberculin  Ruckstand  or   Residual 
Tuberculin. 

The  technique  is  carried  out  by  making  cultures  of  young  tubercle  bacilli  which 
are  then  thoroughly  dried  in  vacuum  and  finely  ground  in  mortars.  The  pulverized 
bacilli  are  agitated  in  distilled  water  and  the  turbid  mass  is  centrifugalized.  The 
sediment  thus  obtained  composes  the  T.  R.  or  the  tubercle  bacilli  residue. 

T.  R.  therefore  contains  the  aqueous  insoluble  components  of  the  tubercle  bacillus, 
while  the  soluble  ones  are  retained  in  the  opalescent  supernatant  fluid  which  Koch  calls 
TO  (Tuberculin  Original). 


58  THE  TUBERCULIN  THERAPY. 

T.  R.  is  readily  assimilated  by  patients.  If  carefully  administered  it  produces  very 
little  infiltration  and  only  slight  temperature  and  general  reaction.  Its  price  is  com- 
paratively high  (i  c.c.  costs  8.50  marks). 

The  first  preparation  which  contained  both  the  soluble  and  insoluble 
elements  of  the  living  bacilli  was  the 

/.  New  Tuberculin — Bacilli  emulsion  (B.  E.)  which  consists  of  T.  R.+ 
T.  O. 

The  living  tubercle  bacilli  are  first  pulverized  in  a  mortar  and  then  suspended  in 
salt  solution.  No  centrifugalization  is  necessary,  but  sedimentation  is  adhered  to,  and 
besides,  50  per  cent,  glycerin  is  added  for  preservation  purposes.  Next  to  T.  the  new 
tuberculin  B.  E.  has  been  most  carefully  studied. 

Equally  lacking  in  being  an  ideal  antigen  is  the  B.  E.  inasmuch  as  immunity  at- 
tained by  its  injections  is  not  at  all  proof  against  subsequent  infection. 

Closely  resembling  the  B.  E.  is 
g.  the  Tuberculin  Beraneck. 

BeVaneck  produced  two  tuberculin  preparations  of  which  one  is  in  the  main  identical 
with  TOA,  while  the  other  is  an  extract  of  tubercle  bacilli  with  i  per  cent  of  phosphoric 
acid.  Both  of  these  tuberculins  are  mixed  together  and  applied.  Sahli  reports  good 
results  with  this  mixture. 

Although  none  of  the  described  tuberculin  preparations  can 

Action  of     be  considered  a  true  antigen  for  the  tubercle  bacillus,  they 

Tuberculin,    have    nevertheless    an    undoubtedly    favorable    effect    upon 

tuberculous  individuals.     To   a  certain  extent   the  benefits 

must  be  said  to  be  derived  by  the  mechanism  of  partial  immunization.     This 

in  itself  does  not,  however,  explain  the  entire  phenomenon  of  their  successful 

action. 

On  examination  of  the  tuberculous  organs  of  animals  treated  with 
tuberculin,  there  will  be  found  within  the  healthy  tissue  surrounding  the 
tuberculous  foci,  a  fresh  inflammatory  reaction.  This  consists  of  a  sero- 
fibrinous  exudate  and  a  zone  of  leucocytes  intruding  to  a  certain  extent  upon 
the  tubercular  lesion.  Tuberculin  acts  only  upon  tuberculous  tissue  which 
is  still  alive  and  not  upon  dead,  cheesy  or  necrotic  structures. 

If  enough  tuberculin  is  given  so  that  death  of  a  tuberculous  guinea-pig  occurs> 
the  changes  found  are  striking.  On  dissection,  about  the  point  of  inoculation  Koch 
reports  a  marked  congestion  of  the  blood  vessels  giving  a  red  and  often  an  almost  dark 
violet  appearance.  This  discoloration  extends  for  a  greater  or  less  distance  from  the 
site  hi  question.  The  neighboring  lymph  glands  are  similarly  reddened.  Besides  the 
tuberculous  changes  present  within  the  liver  and  spleen,  these  organs  show  on  their 
surface  many  blackish-red  spots  varying  in  size  from  that  of  a  pin-point  to  a  hemp  seed, 
and  resembling  very  closely  the  ecchymosis  found  in  some  infectious  diseases.  On 
microscopical  examination  are  found  no  blood  extravasations,  but  very  widely  distended 
capillaries  directly  surrounding  the  tuberculous  foci.  The  capillaries  are  so  densely 
plugged  with  red  blood  cells  that  it  seems  almost  impossible  for  the  circulation  to  have 
continued  in  these  places.  In  exceptional  cases  only,  are  the  blood  vessels  ruptured  and 
the  escaped  blood  found  within  the  tuberculous  foci.  The  lung  presents  similar  changes, 


TECHNIQUE    OF   TUBERCULIN-THERAPY.  59 

but  not  as  regularly  or  of  such  characteristic  appearance.  The  small  intestine  is  often 
deeply  and  evenly  congested.  In  all  this  symptom-complex,  in  short,  the  never  failing 
and  almost  pathognomonic  feature  is  the  hemorrhagic-like  spots  on  the  liver  surface. 

Koch  considered  that  the  tuberculin  brought  about  the  death  of  the 
tuberculous  tissue.  He  furthermore  interpreted  the  disappearance  of  the 
reaction  after  inoculations  with  tuberculin,  as  evidence  that  the  entire  tuber- 
culous structure  had  been  destroyed;  in  other  words  that  healing  had  set  in. 

Accordingly,  in  the  first  tuberculin  era,  the  erroneous  tendency  arose  to 
consider  those  tuberculous  patients  as  cured  who  after  gradually  diminishing 
reactions  to  tuberculin  had  become  entirely  refractory  to  it.  Truth  to  say, 
these  individuals  had  merely  become  immunized  against  old  tuberculin,  and 
had  another  preparation  such  as  new  tuberculin  been  injected,  a  reaction 
would  have  recurred. 

Basing  their  conclusions  on  experimental  work,  Wassermann,  Bruck  and 
also  the  author  have  shown  that  besides  the  factor  of  partial  immunization, 
it  is  the  focal  action  of  the  tuberculin  which  is  the  beneficial  agent  in  its 
therapy. 

The  inflammatory  hyperemia  produced,  leads  to  a  destruction  of  the 
tuberculous  tissue,  while  at  the  same  time  the  inflammatory  process  recedes. 
In  addition  there  is  a  formation  of  connective  tissue  which  encapsules  the 
focus  and  with  it  also,  is  associated  the  local  stimulation  of  antibodies. 

The  Technique  of  Tuberculin -therapy. 

Three  distinct  periods  can  be  noted  in  the  history  of  this  therapy.  The  first  began 
in  the  memorable  year,  1890,  when  Robert  Koch  made  known  his  discovery  of  tuberculin. 
At  this  time,  the  aim  of  tuberculin  treatment  was  to  cause  very  marked  reactions  and 
to  continue  with  the  injections  until  no  further  reaction  was  obtained.  In  lupus,  glandular 
or  bone  tuberculosis  10  mg.  was  the  initial  dose.  In  tuberculosis  of  the  lungs  i  mg.  was 
the  beginning.  If  the  patient  reacted  to  this  amount,  he  received  daily  inoculations  of  this 
dose  until  no  reaction  appeared.  Then  2  mg.  T.  were  given  and  the  same  procedure 
repeated.  Quite  frequently,  depending  upon  the  strength  of  the  individual  concerned, 
10  mg.  was  given  as  the  primary  inoculation  in  phthisis,  and  then  rapidly  increased. 
While  Koch  himself  very  soon  recognized  that  this  rather  severe  treatment  was  suitable 
only  for  incipient  or  moderately  advanced  cases,  very  sick  and  far  advanced  phthisis 
patients  were  similarly  treated  by  many  physicians.  Following  such  procedure,  decidedly 
unfavorable  results  were  obtained  in  the  latter  class  of  patients  and  consequently  a 
marked  waning  in  the  enthusiasm  which  first  greeted  the  tuberculin  therapy  was  the 
inevitable  outcome.  Thus  the  once  highly  praised  remedy  was  entirely  rejected. 

During  the  second  period  only  very  few  former  followers  of  Koch  continued 
their  studies  in  this  field.  These,  however,  made  it  their  business  to  investigate  the 
causes  which  led  to  the  failure  of  tuberculin  therapy.  Their  researches  led  to  new 
principles  in  the  treatment,  and  to  more  exact  knowledge  of  its  indications  as  well  as 
contraindications. 

The  success  obtained  by  the  untiring  efforts  of  these  investigators  brought  about 
after  many  years  a  revival  of  the  interest  in  this  therapy.  It  was  again  taken  up  (third 
tuberculin  era)  and  there  is  no  doubt  that  when  properly  handled,  tuberculin  in  well 


6o 


THE    TUBERCULIN   THERAPY. 


selected  cases  is  of  decided  benefit.  Nevertheless,  even  at  the  present  day,  conclusive 
opinion  as  to  many  of  its  details  cannot  be  formed.  Attempts  are  being  made  to  set 
the  individual  treatment  upon  a  biological  instead  of  upon  the  more  or  less  schematic 
basis  thus  far  employed. 

Old  Tuberculin  (Tuberculin  Koch  T.) 

While  it  was  the  aim  in  the  early  era  of  tuberculin  treatment  to  produce 
very  strong  general  reactions,  it  is  the  general  concensus  of  opinion  at  pres- 
ent that  it  is  best  to  so  arrange  the  tuberculin  therapy  as  to  avoid  a  general 
reaction  and  especially  the  fever.  Gcetsch  was  the  man  who  first  called 
attention  to  this. 

With  such  object  in  view,  one  must  begin  with  small  doses.  Some  men 
start  with  i/ioo  mg.,  others  with  i/io  mg.  T.  If  no  reaction  is  incited, 
the  dose  is  increased  in  five  to  seven  days  to  5/100  mg.  and  then  to  i/io,  2/10, 
4/10,  8/10,  i,  2,  4,  6,  8,  10,  20,  40,  80,  100,  150,  200,  300,  400,  600,  800,  and 
1000  mg.  This  last  amount  represents  the  maximum  dose.  If  the  patient 
still  gives  a  focal  reaction  with  such  a  dose,  it  is  best  to  repeat  it  at  intervals  of 


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CHART  2. — Example  of  Hypersusceptibility  brought  about  by  going  back  to  a  smaller  dose. 

two  to  four  weeks,  until  finally  no  reaction  is  apparent.  Occasionally  one 
will  advance  with  the  doses  at  a  more  rapid  rate,  but  in  general,  inoculations 
should  not  be  repeated  more  than  twice  a  week. 

If  at  any  time  a  distinct  or  even  doubtful  reaction  occurs,  it  is  absolutely 
necessary  to  await  the  complete  subsidence  of  the  latter,  and  then  the  same 
dose  is  to  be  repeated.  In  such  a  case,  an  interval  of  at  least  eight  to  ten 
days  should  elapse.  The  dosage  should  under  no  condition  be  diminished, 
as  thereby  instead  of  immunity,  hyper-susceptibility  is  the  result.  There- 
fore, patients  who  at  a  short  time  previously  had  a  diagnostic  tuberculin 
test  performed  upon  them,  should  receive  the  highest  dose  employed  in  this 
connection  as  the  initial  prescription  for  their  tuberculin  therapy.  The 
following  chart  illustrates  the  condition  of  hyper-susceptibility  occasioned 
by  a  diminution  in  dosage.  (Chart  2.) 


OLD    TUBERCULIN.  6 1 

Patient  had  a  localized  one-sided  apex  tuberculosis.  At  a  diagnostic  tuberculin 
injection,  he  reacted  only  when  o.oi  c.  cm.  T.  was  employed.  After  the  interval  of  a 
month  the  patient  was  advised  tuberculin  treatment.  Contrary  to  the  rule  just  cited, 
he  received  as  a  first  injection  not  o.oi  tuberculin  but  0.002  c.cm.  T.  With  this  small 
dose  he  already  had  an  increase  of  temperature,  although  coming  rather  late,  and  not 
quite  typical.  After  this  reaction  had  disappeared,  without  any  other  manifestations,  the 
same  dose  of  o.  002  c.  cm.  tuberculin  was  repeated  and  as  evident  from  the  chart,  a  very 
marked  response  was  inaugurated.  This  was  accompanied  by  a  chill,  vomiting,  head- 
ache, general  pains  and  weakness.  In  addition  there  was  a  slight  relapse  after  the 
aforementioned  symptoms  had  disappeared.  In  order  to  immunize  this  patient  against 
his  hyper-susceptibility,  it  was  advisable  to  repeat  the  dose  of  0.002  c.cm.  T.  at  which 
the  reaction  reappeared,  but  in  a  very  much  milder  form.  It  was  only  after  the  fifth 
inoculation  of  the  same  dose  that  no  reaction  was  in  evidence.  Thus  was  the  hyper- 
susceptibility  overcome  and  the  patient  treated  in  the  general  way. 

The  danger  of  hyper-sensitiveness  also  exists  if  the  same  reactionless 
dose  is  too  frequently  repeated;  especially  so  if  the  quantities  injected  are 
small.  The  higher  the  dosage,  the  less  liable  is  the  occurrence  of  hyper- 
susceptibility. 

This  question  is  above  all  to  be  considered  when  after  a  certain  interval, 
a  second  course  in  tuberculin  therapy  is  advised.  In  general  it  can  be  car- 
ried out  after  a  period  of  three  months,  even  though  sometimes  certain 
difficulties  may  be  met  with.  Petruschky  strongly  recommended  this 
treatment,  in  successive  stages.  (Etappenbehandlung) .  The  author  is  of 
the  opinion  that  it  is  best  to  retain  the  patient  as  long  as  possible  at  his 
acquired  immunity  (tuberculin)  by  stretching  the  course  of  treatment  over  a 
long  period  of  time.  He  therefore  repeats  an  inoculation  of  the  maximum 
dose,  every  three  or  four  weeks  and  when  hyper-susceptibility  arises,  he 
changes  the  preparation  and  begins  with  a  small  dose  again. 

As  for  the  technical  details  of  the  treatment,  several  practical  suggestions 
may  be  made. 

1.  The  inoculation  should,  if  possible,  be  given  in  the  morning  hours, 
for  a  restless  night  usually  follows  an  injection  in  the  evening. 

2.  It  is  best  to  so  arrange  the  dilutions  that  the  patient  receives  a  frac- 
tion of  i  c.cm.  at  each  injection. 

3.  The  site  of  injection  should  be  alternated  between  the  back  and  the 
breast. 

4.  The  temperature  should  be  taken  every  two  or  three  hours  and  a 
chart  of  the  same  kept. 

5.  Disturbances  in  the  general  condition  of  the  patient  without  the 
presence  of  fever  are  to  be  considered  in  the  light  of  general  reactions  just  as 
fever  without  other  disturbances. 

6.  The  patient's  weight  should  be  taken  regularly  every  week,  and  then 
the  dose  should  be  increased  provided  no  loss  in  weight  has  taken  place. 

7.  In  cases  where  the  pulse  increases  in  rate  or  becomes  poorer  in  quality, 


62 


THE  TUBERCULIN  THERAPY. 


treatment 

ning, 

cases 


the  treatment  should  be  undertaken  very  carefully  and  the  pulse  constantly 

kept  as  guide.      Slowness  of  pulse  can,  as  a 
rule,  be  considered  a  signum  bonum. 

Especially    favorable    for    the    tuberculin 
are   the  individuals  with  a  begin- 
localized    pulmonary    tuberculosis,    or 
of   lupus,   and  renal   tuberculosis,   as 
reported  by  Lenhartz.     The  presence  of  fever 
leads   some  to  consider  such  application  as 
contraindicated.     This  is  indeed  incorrect,  as 
frequently  it  is  observed  that  a  chronic  fever 
entirely  disappears  during  a  course  of  treat- 
ment, and  very  often  remains  away.     Even 
if  the  fever  continues,  a  good  result  in  the 
general  condition  of  the  patient  is  neverthe- 
less obtained.     (Chart  3.) 

Patient  H. — Ninteen  years  old  with  distinct  tuber- 
culous habitus,  on  admission  to  the  medical  service, 
presented  a  marked  infiltration  and  catarrh  of  two- 
thirds  of  the  right  lung  with  a  cavity  in  the  upper 
lobe;  infiltration  of  the  left  lobe  and  a  great  number 
of  tubercle  bacilli  in  the  sputum;  marked  weakness 
and  continuous  fever.  In  five  weeks  the  patient  had 
gained  n  kg.  in  weight; — 8  kg.  in  one  week. 

Simultaneously  his  general  condition  improved 
very  much;  the  night  sweats  disappeared,  and  the 
cough  diminished,  but  the  number  of  bacilli  still  re- 
mained the  same,  and  the  physical  signs  of  the  lungs 
unaltered.  Subsequently  the  patient  received  treat- 
ment also  with  B.E.,  with  the  consequence  that  the 
temperature  finally  subsided,  the  cough  and  sputum 
likewise,  and  the  bacilli  became  few  and  at  times 
entirely  absent  for  several  days  in  succession.  In 
fact,  the  general  condition  became  excellent.  Objec- 
tively, there  was  no  demonstration  of  catarrh al 
affection. 

In  this  connection  it  might  be  noted  that 
such  a  remarkable  increase  in  weight  in  so 
short  a  time  is  by  no  means  the  rule,  although 
good  effects  are  observed  in  many  cases. 

Naturally  the  medical  treatment  should 
not  be  limited  to  the  tuberculin  therapy.  If 
even  in  the  immunization  of  healthy  animals, 
attention  is  paid  to  their  housing  and  feeding, 
how  much  more  imperative  is  this  considera- 
tion when  applied  to  sick  human  individuals. 


- 
•c 


NEW    TUBERCULIN. 


Bearing  in  mind  that  just  as  in  any  other  treatment,  success  is  only 
achieved  by  creating  a  favorable  medium,  the  same  focussing  of  good 
influences  should  be  employed  in  tuberculin  therapy.  Rest  and  forced 
feeding  are  curative  factors  which  one  cannot  neglect,  and  the  best  places 
for  the  obtention  of  these,  at  the  beginning  at  least,  are  hospitals,  sanatoriums 
or  convalescent  homes.  When  in  such  a  way,  the  general  status  of  the  pa- 
tient is  improved,  ambulant  therapy  can  be  continued. 

As  for  the  contraindications  to  tuberculin  treatment,  it  is  very 
Contraindica-  difficult  to  set  general  rules.     The  opinions  of  various  authori- 

tions  to      ties  differ  greatly  on  the  subject.     While  for  example,  Moller 
Tuberculin    and  others  consider  hemoptosis  as  a  distinct  contraindication, 

Therapy.  Aufrecht  and  Kramer  claim  that  under  tuberculin  therapy 
hemoptosis  is  decidedly  improved.  It  is  easy  to  understand 
this  difference  in  attitude,  if  the  changes  in  the  focal  reaction  are  considered. 
There  is  no  doubt  that  hemoptosis  may  be  excited  by  increased  supply  of 
blood  and  the  inflammatory  process  associated  with  the  inoculation  of  tuber- 
culin. The  more  severe  the  focal  reaction,  the  greater  is  this  possibility. 
On  the  other  hand,  the  new  formation  of  connective  tissue  and  the  absorp- 
tion of  the  tuberculous  tissue  will  diminish  the  frequency  of  hemorrhage. 
With  a  general  tendency  toward  hemoptosis,  it  is  therefore  best  to  wait  a 
long  time  after  the  cessation  of  the  latter,  and  then  begin  with  small  doses. 
The  patient  should  be  under  careful  observation  and  by  constant  physical 
examination,  any  possible  focal  reaction  should  be  controlled.  If,  in  spite 
of  this,  hemoptosis  does  set  in,  one  should  not  at  once  be  discouraged.  An 
interval  of  about  fourteen  days  is  to  be  allowed  to  pass,  and  then  the  treat- 
ment again  undertaken.  Frequently,  the  hemoptosis  will  cease.  If  not, 
or  if  the  patient  loses  in  weight  and  becomes  weaker,  the  tuberculin  therapy 
should  be  discontinued. 

As  further  contraindications,  Moller  mentions  marked  general  weak- 
ness, fever,  heart  affections,  epilepsy,  and  hysteria.  In  full  agreement  with 
Bandelier  and  Roepke,  the  author  does  not  consider  any  of  the  above  as 
cause  for  the  non-employment  of  tuberculin.  Only  where  absolute  cachexia, 
without  any  possibility  for  improvement  exists,  is  this  therapy  to  be  omitted. 
In  all  other  conditions,  an  attempt  is  by  all  means  justified.  Experience, 
as  a  matter  of  course,  plays  an  important  role  in  the  selection  of  suitable 
cases.  •  For  a  beginner,  it  is  advisable  to  gain  practice  by  the  treatment  of 
uncomplicated  cases  before  undertaking  those  of  greater  difficulty. 
2.  New  Tuberculin,  Bacilli -Emulsion  (B.  E.)  and  New  Tuberculin  T.  R. 

Treatment  with  new  tuberculin  follows  along  the  very  same  lines  set 
down  for  old  tuberculin. 

New  tuberculin  T.  R.  is  the  mildest  of  all  preparations.  It  is  very 
suitable  for  the  beginning  treatment  of  susceptible  patients.  When  the  in- 
dividual does  not  react  to  large  doses,  it  is  well  to  start  in  with  B.  E.  The 


64 


THE    TUBERCULIN   THERAPY. 


employment  of  B.  E.  can  also  be  affected  without  producing  any  reaction, 
although  this  is  somewhat  more  difficult. 

The  dosage  scheme  advised  by  Bandelier  and  Roepke  is  as  follows: 

i/iooo,  2/1000,  3/1000,  7/1000,  10/1000  mg., 

15/1000,  2/100,  3/100,  5/100,  7/100,  10/100  mg., 
At  intervals  of  i  to  2  days; 
15/100,  2/10,  3/10,  5/10,  7/10,  10/10  mg., 

At  intervals  of  2  to  3  days; 
12/10,  15/10,  2,  2  1/2,  3,  mg., 

At  intervals  of  3  to  4  days; 

4,  5,  6,  7,  8,  9,  10  mg., 
at  4  to  6  to  10  days  intervals. 

In  susceptible  patients,  it  is  best  to  increase  the  dosage  only  by  one-half 
mg.  even  when  large  doses  are  administered.  Ten  mg.  B.  E.  represents 
the  maximal  dose. 

The  author  himself  follows  a  different  scheme  from  that  of  Bandelier 
and  Roepke.  The  injections  are  given  less  frequently,  only  about  once  a 
week,  but  the  dose  is  always  increased  twofold,  fivefold  and  even  tenfold 
without  any  excessive  reactions. 

Fever  is  obtained  much  less  often  with  new  tuberculin  than  with  T.  The 
reaction  usually  is  in  the  form  of  lassitude,  nausea,  weakness,  insomnia,  etc. 

The  treatment  with  new  tuberculin  is  particularly  favorable  in  cases 
where  a  low  continuous  fever  is  present.  It  also  is  more  potent  in  destroying 
the  bacilli  of  the  sputum.  The  author  therefore  prefers  this,  especially  the 
B.  E.  to  all  other  tuberculin  preparations.  Bandelier  and  Rcepke  have  also 
obtained  gratifying  results  with  the  B.  E.  therapy  as  is  evident  from  the 
following  statistics  of  205  patients  treated  at  the  sanatorium  at  Kottbus. 


Stage. 

I. 

IT. 

III. 

Cured  (A)   .  . 

23  —  II     22% 

JO  —     77    O4% 

I?  =  IO   48% 

o  =  o       % 

Completely  able  bodied  (BI).  .  . 

98=47.80% 

12=  44-44% 

77=62.09% 

9  =  l6.63% 

Satisfactory  result  (A  +  BI)  
Able   bodied   in   terms  of  law 
(BIT). 

121=59.02% 
70  =  34.15% 

22=   81.48% 
5=   18.52% 

90  =  72.57% 
30  =  24.19% 

9  =  16.63% 

35=64.81% 

Total  improvement   (A  +  BI  + 
BII). 
Negative  result  (C) 

I9I=93.I7% 

14  —  6  83% 

27=100       % 

120=96.76% 

4  =    "?    24.% 

44=81.44% 

10  —  18  56% 

Bacilli  in  sputum  on  admission. 

114  =  55.61% 

4=    14.81% 

63=50.81% 

47=87.04% 

Absence  of  bacilli  or  sputum  at 
discharge. 

59=51.78% 

4  =  100       % 

49=77.78% 

16=34.34% 

TUBERCULOUS    SERUM. 


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fact  that  sensitization  of  bacteria 
tends  to  neutralize  those  substances 
which  produce  infiltrations.  This 
last  has  been  demonstrated  by  the 
author  in  cases  of  mouse-typhoid, 
and  swine  pest  bacilli,  where  marked 
infiltrations  following  their  inocula- 
tion have  by  afore-mentioned  means 
of  sensitization  been  avoided.  The 
following  chart  illustrates  prelimi- 
nary treatment  with  S.  B.  E.  followed 
by  B.  E.  (Chart  4.) 

The  patient  was  a  female  who  at  the 
time  of  admission  presented  double-sided 
apical  pulmonary  and  suspicious  intestinal 
tuberculosis.  Tubercle  bacilli  were  present 
in  the  sputum.  The  patient  was  dis- 
charged from  the  clinic  as  relatively  cured, 
i.e.,  all  manifestations  of  illness  had  disap- 
peared with  the  exception  of  slight  dulness 
over  one  of  the  apices  which,  however,  could  have  been  attributed  to  cicatrization. 
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In  respect  to  the  objection  frequently  advanced  that  B.  E.  is  absorbed 
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that  this  may  be  readily  overcome  by 
preliminary  use  of  T.  R.  or  by  the  em- 
ployment of  sensitized  B.  E.,  as  has 
been  advised  by  Meyer  and  Rupple. 

By  sensitized  B.  E.  is  under- 
stood, a  bacilli  emulsion  which  has 
been  mixed  with  the  tuberculous 
serum  of  a  horse  or  ox  containing 
anti-tuberculin.  This  mixture  brings 
about  a  union  between  certain  of  the 
antibodies  and  substances  contained 
within  the  bacteria.  The  tuberculous 
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fugalization  and  washing  of  the  mix- 
ture with  physiological  salt  solution. 

The  sensitized  B.  E.  (S.  B.  E.)  is 
milder  than  B.  E.  and  in  its  character 
otherwise  more  like  T.  R.     The 


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66  THE  TUBERCULIN  THERAPY. 

In  addition,  there  was  normal  vesicular  breathing,  no  temperature,  no  catarrh,  and  a 
good  general  condition.  Undoubtedly  it  is  difficult  to  say  whether  this  case  is  cured. 
Only  years  of  observation  can  prove  this.  A  temporary  latency  of  symptoms  must 
always  be  considered.  Suffice  it  to  say,  that  the  patient  was  a  great  deal  improved 
and  able  to  return  to  her  work. 

On  close  observation  of  this  chart  it  will  be  noticed  that  practically  no 
reactions  occurred  in  spite  of  the  rather  rapid  increase  in  the  dosage.  Slightly 
increased  temperatures  were  manifest  only  occasionally  (o.ooooi  c.c.  S. 
B.  E.).  Even  though  the  same  dose  was  not  repeated  on  account  of  the 
long  intervals  between  the  individual  injections,  no  increased  reaction 
appeared  after  subsequent  inoculation.  When  o.i  c.c.  of  S.  B.  E.  produced 
no  reaction,  the  susceptibility  to  B.  E.  was  tested.  Doses  of  o.oooi  B.  E.  to 
0.5  B.  E.  were  administered  at  short  intervals,  without  any  symptoms. 
Only  after  i.o  c.c.  of  B.  E.,  was  there  a  slight  increase  of  temperature  with 
rather  marked  general  manifestations — (headache,  pains  in  the  extremities, 
weakness  etc.)  which  subsided  within  twenty-four  hours.  On  repetition  of 
the  same  dose,  no  reaction  occurred. 

Six  days  later,  when  for  the  third  time  i  c.c.  of  B.  E.  was  given  there 
appeared  a  very  marked  disturbance  throughout  the  system,  an  evidence  of 
hyper-susceptibility.  (See  Chart  2). 

3.  Bo  vine -tuberculin. 

Koch's  differentiation  between  bovine  and  human  tuberculosis  led  to  the 
attempt  at  immunization  of  cattle  with  human  tubercle  bacilli.  (Bovo- 
vaccine  of  Behring,  and  Tauruman  of  Koch). 

Spengler  tried  to  reverse  this  use  and  employ  the  milder,  infectious 
bovine  bacilli  for  the  tuberculin  therapy  in  man.  He  used  these  bacteria  to 
make  up  preparations  similar  to  the  old  and  new  tuberculin.  In  this  connec- 
tion he  had  recourse  especially  to  the  P.  T.  O.  (Perlsucht  Original  Tuber- 
culin) i.e.,  the  preparation  analogous  to  T.  O.  A. 

Bovine  tuberculin  is  said  to  be  borne  better  than  the  human.  The 
reactions  are  supposed  to  be  of  a  nature  less  severe,  and  the  therapeutic 
results  just  as  good  or  even  better. 

4.  Nastin. 

All  the  above  mentioned  preparations  have  as  their  aim  the  production  of  an  inocu- 
lation material  which  is  to  contain  the  substances  embodied  within  the  tubercle  bacilli, 
and  which  are  more  or  less  correctly  considered  as  representing  their  poisonous  elements. 
Deycke  and  Reschad  showed  that  the  fat-like  material  encapsulating  the  bacteria  to 
which  is  ascribed  their  strongly  acid  fast  character,  also  plays  an  important  r61e  in  the 
question  of  tuberculosis  immunity.  These  men  prepared  a  wax-like  substance,  nastin, 
from  a  streptothrix  which  they  found  as  a  saprophyte  in  a  case  of  leprosy — streptothrix 
leproides.  Nastin  closely  resembles  the  fat-like  substance  of  the  tubercle  bacilli  and 
with  it  one  can  immunize  healthy  guinea-pigs  against  living  virulent  tubercle  bacilli. 


NASTIN.  67 

In  the  treatment  of  tuberculosis,  however,  it  has  no  beneficial  effect.  On  the  day  after 
inoculation,  fever  sets  in,  sputum  increases  in  great  quantities  and  contains  large  amounts 
of  tubercle  bacilli.  In  leprosy,  slight  improvement  has  been  noticed  by  its  use. 

Metalnikoff  has  confirmed  the  above  findings  and  further  shown  that  the  bee  moth, 
Galeria  Molinella,  attributes  its  very  high  immunity  against  tuberculosis  to  a  strong  wax 
dissolving  ferment  possessed  by  it.  It  is  probable,  too,  that  inoculations  of  nastin  there- 
fore, produce  antibodies  which  have  the  power  of  dissolving  fat.  In  this  way  the  capsule 
of  the  tubercle  bacillus  is  destroyed  and  the  antigen  is  liberated  to  be  absorbed.  While 
healthy  animals  can  thus  be  immunized,  tuberculous  individuals  would  be  indirectly 
receiving  a  tuberculin  injection,  and  its  amount  would  depend  upon  the  quantity  of 
tubercle  substance  suddenly  liberated.  It  seems  to  the  author  that  the  more  rational 
way  of  conducting  this  therapy  would  be,  to  first  obtain  a  high  immunity  against  the 
substance  of  the  tubercle  bacillus  by  injection  with  B.E.,  and  then  to  follow  this  by  treat- 
ment with  nastin.  Such  "fractional"  treatment  may  prove  an  interesting  new  step  in 
tuberculin  therapy. 


CHAPTER  VII. 
TOXIN  AND  ANTITOXIN. 

So  far,  the  preceding  chapters  have  dealt  with  immunization  by  the 
bacterial  bodies  and  substances  extracted  from  them.  Further  attention 
must,  however,  be  paid  to  the  products  of  secretion  of  bacteria,  namely  the 
toxins.  Only  few  classes  of  bacteria  have  true  soluble  toxins  such  as  are 
possessed  by  tetanus  and  diphtheria  bacilli.  The  symptom-complex  incited 
by  the  toxin  producing  bacteria  differs  decidedly  from  that  of  the  sepsis 
class. 

A  comparison  between  anthrax  and  tetanus  certainly  exhibits  a  striking 
difference.  Although  both  are  wound  infections  incited  by  characteristic 
bacteria,  smears  of  the  pus  from  wounds,  in  the  case  of  anthrax,  display  on 
examination  numerous  bacilli,  while  in  the  case  of  tetanus,  the  bacillus  is 
very  sparsely  found.  Even  carefully  prepared  anerobic  cultures,  or  inocu- 
lations in  mice  of  the  pus  itself,  do  not  always  successfully  demonstrate  the 
presence  of  the  tetanus  bacillus.  In  the  blood,  lymph  glands  and  viscera 
of  anthrax  cases,  excessively  large  numbers  of  microbes  can  be  exposed, 
while  even  in  the  most  fatal  cases  of  tetanus,  there  is  nowhere  any  evidence 
of  bacteria  or  their  spores.  Where  so  many  living  foreign  organisms  are 
found  invading  the  individual,  no  hypotheses  are  necessary  for  explanation 
of  the  associated  marked  disturbances  as  in  anthrax;  it  is,  however,  more 
complex  to  understand  the  severity  of  the  symptoms  in  conditions  like 
tetanus,  where  such  exceedingly  scant  bacteriological  findings  exist.  Here 
the  micro-organisms  play  only  a  secondary  r61e,  the  entire  symptom-com- 
plex being  produced  by  a  poison  extruded  from  the  bacteria.  In  diphtheria, 
conditions  are  similar  to  those  in  tetanus,  although  in  the  former  the  bacilli 
can  be  readily  demonstrated  both  microscopically  and  by  culture.  Even 
though,  however,  the  localization  of  bacteria  in  diphtheria  is  confined  to 
organs  not  absolutely  essential  for  life — diseased  tonsils — these  themselves 
do  not  explain  the  alarming  situation  observed  in  this  disease;  for  the  real 
cause  of  the  illness  is  to  be  found  in  the  toxin  which  is  secreted  by  the 
bacteria  localized  in  them,  and  distributed  by  the  blood  stream  throughout 
the  entire  system. 

That  a  toxin  really  exists,  and  is  not  hypothetical,  Roux  and  Yersin,  as 
well  as  Kitasato  have  proven  by  demonstration  of  the  poisonous  agents  in 

68 


ACTION    OF    DIPHTHERIA   TOXIN.  69 

the  bouillon  culture  of  both  diphtheria  and  tetanus.  As  most  cultures  show 
only  slight  tendencies  to  toxin  formation,  the  evidence  of  a  virulent  toxin 
may  necessitate  a  special  strain  of  the  bacterium. 

The  length  of  time  required  by  cultures  for  the  production  of  moderate 
amounts  of  toxins  is  by  no  means  constant.  With  diphtheria  this  varies 
from  several  days  to  2  to  3  weeks.  As  a  general  rule,  if  toxin  is  hot  liberated 
within  the  first  four  weeks  it  will  most  probably  not  appear  after  that  time. 
On  its  appearance,  it  is  isolated  by  filtering  the  bouillon  culture  first  through 
filter  paper  to  remove  the  pellicle,  and  then  through  a  bacterial  filter  to  get 
rid  of  the  bacteria.  A  layer  of  toluol  i  to  2  cm.  is  added  for  the  purposes 
of  sterilization  and  it  is  advisable  to  agitate  the  toxin  and  toluol  thoroughly 
every  day  to  prevent  contamination. 

It  does  not  fall  within  the  scope  of  this  book  to  take  up  the  various 
methods  proposed  for  obtaining  and  preserving  the  various  toxins.  It  is 
the  object  merely  to  review  the  details  associated  with  their  mode  of  action 
and  standardization. 

The  first  and  most  important  member  of  this  group  is  the  diphtheria 
toxin. 

The  diphtheria  toxin  is  first  tested  by  subcutaneous  injections  into 
Action  of      guinea-pigs  250  mg.  in  weight.     The  action  of  the  toxin  is  entirely  de- 
Diphtheria     pendent  upon  the  dosage;  the  more  toxin  injected  the  more  rapidly  does 
Toxin.        death  occur.     This,  however,  is  not  to  be  taken  in  mathematically  correct 
proportions — i.e.,  twice  the  dose  does  not  produce  the  same  action  in 
one-half  the  time.     A  certain  period  of  time  must  always  elapse  before  death  can  take  place,  the 
minimum  being  about  one  day.     The  interim  is  known  as  the  period  of  incubation  and 
it  is  the  existence  of  this  that  goes  to  make  one  of  the  essential  characteristics  of  a  true 
toxin.     A  toxin  requires  a  definite  period  of  time  for  its  action  to  become  manifest;  and 
even  the  largest  dose  of  toxin  cannot  diminish  the  length  of  this  period  below  a  certain 
minimum.     On  the  other  hand,  the  length  of  the  incubation  time  can  be  increased  by  the 
injection  of  a  smaller  dose,  so  that  ultimately  a  dose  small  enough  is  obtained  which  is 
not  instrumental  in  producing  death  (Dosis  subletalis). 

If  a  guinea-pig  is  inoculated  with  a  quantity  of  toxin  sufficient  to  kill  it  in  three  to  four 
days,  nothing  abnormal  is  evident  the  first  day;  various  manifestations  of  illness,  however, 
follow  soon  after. 

Edema  appears  at  the  site  of  inoculation.  The  animal  stops  eating,  sits  in  a  corner, 
and  reacts  poorly  to  sound.  Gradually  it  becomes  weaker,  so  that  when  placed  upon  its 
back  it  does  not  resume  its  normal  position;  the  temperature  which  at  first  rose  some- 
what, falls  abruptly  and  then  death  takes  place. 

At  autopsy,  a  gelatinous  and  strongly  hemorrhagic  edema  is  found  which  starts  at 
the  site  of  the  injection.  On  opening  the  abdominal  cavity  one  finds  but  very  little  peri- 
toneal exudate,  strongly  injected  vessels  of  the  mesentery,  and  especially  characteristic, 
markedly  reddened  adrenal  glands.  In -the  thorax  are  found  bloody  pericardial  and 
pleural  exudates,  and  consolidated  areas  in  the  lungs. 

After  the  injection  of  smaller  doses,  edema  likewise  arises  and  becomes  larger  in 
extent  the  slower  the  case  progresses.  Besides  this,  the  animal  loses  in  weight.  With 
sublethal  doses,  edema  or  infiltration  is  confined  to  the  site  of  injection,  and  finally,  with 
the  minutest  doses,  no  edema  occurs,  but  the  hair  falls  out  at  the  place  of  injection. 


70  TOXIN  AND   ANTITOXIN. 

Guinea-pigs  surviving  a  dose  of  toxin,  may  after  two  to  four  weeks  begin  to  show 
paresis  first  of  the  hind,  then  of  the  fore  extremities,  and  finally  even  of  the  muscles  of 
the  back  and  respiration.  The  most  severe  types  of  such  conditions,  however,  may  fully 
subside.  They  may  be  considered  as  analogous  to  the  post-diphtheritic  paralysis  taking 
place  in  man,  which  is  usually,  as  is  well  known,  of  a  benign  nature. 

Besides  guinea-pigs  other  animals  suitable  for  diphtheria  experimental  work 
are  rabbits  (especially  by  intravenous  injection)  and  pigeons  (by  intramuscular 
injection). 

The  susceptibility  of  animals  towards  diphtheria  toxin  varies  greatly,  as  is  seen  from 
the  following  scale  of  Behring,  the  least  susceptible  animals  being  mentioned  first: 
mouse,  rat,  dog,  guinea-pig,  rabbit,  sheep,  cow,  horse,  goat. 


The  strength  of  the  diphtheria  toxin  is  estimated  as  follows: 

Estimation  of  Guinea-pigs  of  equal  size  (250  gms.)  receive  subcutaneous 

Strength  of    injections   of  decreasing   amounts  of  toxin.     With  a  strong 

Diphtheria    toxin,  centi-  and  milligrams   or  even  smaller  denominations 

Toxin.       are  Of  sufficient  potency  to  produce  death.     Doses  such  as 

these  are  not  injected  unless  diluted  in  normal  salt  solution. 

For  exact  results  one  must  not  depend  upon  the  findings  brought  out  by 

the  injection  of  a  single  animal  with   each  dilution;  several  should  be 

inoculated  with  the  same  dose  and  the  effects,  which  should  be  the  same  in 

all  cases,  noted.     It  is  impossible  to  state  beforehand  how  many  dilutions 

may  be  necessary.     If  the  various  actions,  dependent  upon  the  successive 

gradations  of  dosage  are  successfully  represented,  the  experiment  may  be 

taken  as  conclusive;  that  is  to  say,  the  smallest  doses  must  leave  the  animal 

entirely  unaffected,  the  moderate  produce  slight  local  and  general  symptoms, 

and  the  larger  ones  cause  death  of  the  animals.     If  it  should  so  happen 

that  they  all  die,  a  new  set  of  experiments  employing  a  lower  scale  of  dosage 

should  be  undertaken. 

Thus  it  is  seen  that  the  action  of  diphtheria  toxin  is  subject  to  the  quantity 
of  the  toxin  injected.  If  several  different  diphtheria  toxins  are  tested  at  the 
same  time,  it  is  at  once  evident  what  far  reaching  differences  may  arise. 
While  o.ooi  c.c.  of  one  diphtheria  toxin  kills  a  guinea-pig  in  twenty-four 
hours,  a  different  diphtheria  toxin  performs  the  same  action  with  a  dose  ten 
times  as  great,  e.g.,  o.oi  c.c.  The  second  toxin  thus  contains  only  one- 
tenth  as  many  of  the  active  substances.  In  order  to  obtain  a  uniform  method 
for  estimating  the  strength  of  a  diphtheria  toxin  and  thus  get  comparative 
values,  a  standard  unit  of  the  same  has  been  adopted.  And  this  consists  of  the 
smallest  amount  of  toxin  that  will  kill  a  healthy  guinea-pig  weighing  about  250 
gms.  in  four  to  five  days.  This  is  known  as  the  minimum  lethal  dose  or  dosis 
letalis  minima.  In  addition  to  this  "direct  toxic  value,"  it  is  frequently 
important,  especially  for  the  standardization  of  curative  sera,  to  estimate 
the  "indirect  toxic  value"  by  which  is  meant  the  amount  of  antitoxin  which 
a  toxin  can  bind  or  neutralize. 


ANTITOXIN.  7! 

If  an  animal,  e.g.,  a  goat  is  injected  with  a  sublethal  dose  of 
Active  Immu-  diphtheria  toxin  and  after  the  lapse  of  a  certain  period  of  time 
nization      it  is  reinjected  with  a  lethal  dose,  the  animal  remains  alive. 
against       In  fact  it  may  receive  numerous  fatal  doses,  and  still  survive, 
a  Toxin.      ^his    experiment   is    the    simplest    in    active   immunization 
against  a  toxin.     The  explanation  of  the  action  which  has 
taken  place,  an  examination  of  the  blood  serum  of  the  immunized  animal 
will  disclose  very  readily.     If  this  serum  is  mixed  with  a  fatal  dose  of  toxin 
and  the  mixture  inoculated  into  a  normal  guinea-pig,  the  latter  remains  alive 
and  perfectly  active.     The  serum  of  the  immunized  animal 
Antitoxin,     therefore  contains  a  protective  agent  which  is  directed  against 
the  toxin  and  destroys  its  activity;  hence  the  name  antitoxin. 
But  the  antitoxin  is  specific,  i.e.,  diphtheria  antitoxin  neutralizes  only  diph- 
theria toxin  and  not  tetanus.     The  recognition  of  these  facts  and  those 
heretofore  mentioned,  and  the  recommendation  of  the  therapeutic  use  of 
diphtheria  serum  belongs  entirely  to  v.  Behring  and  righteously  may  he  be 
called  the  father  of  serum  therapy. 


Although  theoretically,  the  serum  of  any  animal  immunized  with  diphtheria  toxin  can 
serve  as  a  curative  serum  for  diphtheria,  practical  experience  has  taught  that  it  is  best 
to  employ  horses  for  this  purpose.  For  laboratory  experiments  goats  should  be  the  ani- 
mals of  choice.  It  is  advisable  to  use  the  above  animals  for  the  reason  that  larger  quanti- 
ties of  serum  are  obtained  and  furthermore  because  it  has  been  found  impossible  to  immu- 
nize guinea-pigs  with  previously  unchanged  diphtheria  toxin  even  if  the  initial  dosage  used 
is  the  smallest  subdivision  of  the  minimal  lethal  dose.  Behring  and  Kitashima  showed  that 
after  repeated  injections  of  very  minute  doses  they  were  able  to  kill  guinea-pigs  even 
with  1/400  of  the  dosis  letalis  minima.  This  is  but  another  example  of  an  effect  just 
opposite  to  that  of  immunity  and  known  as  hyper -susceptibility  or  hypersensitiveness,  which 
has  already  been  described  in  the  chapter  on  tuberculin  therapy.  If,  however,  it  is  de- 
sired to  immunize  guinea-pigs,  a  modified  form  of  the  diphtheria  toxin  must  be  employed 
for  the  first  injections.  Several  modifications  are  feasible.  Behring  and  Kitasato  added 
iodin  trichlorid  to  the  toxin  while  Roux  and  Martin,  LugoPs  solution;  C.  Frankel  heated 
it  to  60°,  and  Behring  advocated  the  so-called  " simultaneous  method"  (of  special  aid  in 
tetanus  toxin),  where  mixtures  of  toxin  and  antitoxin  are  injected  and  gradually  the 
quotient  of  the  latter  is  diminished  until  finally  it  is  entirely  omitted.  If  the  animals  have 
borne  the  first  inoculations  of  the  modified  toxin  without  any  ill  effects,  one  may  then 
devote  himself  to  the  use  of  the  unmodified  toxin. 

In  contrast  to  small  animals,  horses  can  be  immunized  with  unmodified  diphtheria 
toxin  right  from  the  start.  Nevertheless  great  care  must  here  also  be  exercised.  Certain 
it  is,  that  less  risk  is  run  in  the  employment,  with  even  the  larger  animals,  of  a  modified 
toxin.  For  the  production  of  a  good  diphtheria  serum,  healthy  horses  about  five  to  six 
years  old  are  used  and  into  them  gradually  increasing  amounts  of  diphtheria  toxin  are 
injected  subcutaneously  or  even  intravenously;  thus  agreeing  with  Ehrlich's  findings 
to  the  effect  that  the  antitoxin  content  of  a  serum  can  be  raised  by  successively  increasing 
the  amount  of  toxin  injected.  As  far  as  the  efficiency  of  the  serum  produced  is  concerned, 
it  is  entirely  dependent  upon  the  animal.  Horses  vary  greatly  in  their  individual  pre- 
disposition towards  the  making  of  an  effective  serum;  some  animals  even  completely 


TOXIN  AND  ANTITOXIN. 


fail  to  do  so,  not  that  the  latter  are  not  actively  immunized,  for  they  are,  but  because  they 
contain  very  little  antitoxin  within  their  serum. 

It  is  impossible  to  recommend  a  distinct  scheme  for  the  immunization  of 
a  horse.  The  intervals  between  the  injections  and  the  size  of  the  dose  is 
entirely  dependent  upon  the  reaction  of  the  animal  toward  previous  inocu- 
lations. A  good  rule  to  follow  is,  that  a  fresh  injection  should  be  given  only  if 
the  reaction  from  the  preceding  one  has  entirely  subsided.  The  reactions  are 
both  local  and  general.  The  local  reaction  comes  in  the  form  of  edema, 
infiltration,  and  sterile  abscesses;  the  general,  loss  in  weight  and  appetite 
and  increase  in  temperature. 

The  following  chart  of  Salomonsen  and  Madsen,  of  the  Copenhagen  Serum  Institute, 
serves  as  an  example  of  how  such  a  diphtheria  serum  is  produced.  A  gravid  mare 
665  kg.  in  weight  was  selected  and  injections  were  given  as  follows. 


Day. 

Dose  of 
Toxin. 

Remarks. 

Day. 

Dose  of 
Toxin. 

Remarks. 

i  cm. 

135 

Serum  removed  contained 

6 

i  cm. 

150  immunity  units. 

12 

3  cm- 

154 



Birth  of  colt. 

_- 

5  cm. 

177 

Serum  removed  contained 

0 

-1/  / 

23 

10  cm. 

45  immunity  units. 

27 

20  cm. 

184 

100  cm. 

36 

25  cm. 

188 

200  cm. 

41 

50  cm. 

J95 

400  cm. 

45 

75  cm- 

205 

700  cm. 

5° 

ico  cm. 

• 

213 

800  cm. 

57 

150  cm. 

223 

600  cm. 

72 

250  cm. 

232 

600  cm. 

81 

450  cm. 

242 

1000  cm. 

02 

600  cm. 

2^2 

Serum  removed  contained 

y 
104 

800  cm. 

1  20  immunity  units. 

119 

1000  cm. 

The  time  selected  for  venesection  is  important.  Antitoxins  like  any 
other  antibodies  do  not  arise  immediately  after  an  injection,  but  only  after  a 
certain  incubation  period.  The  amount  of  antitoxin  first  gradually  increases, 
then  begins  to  sink,  and  after  that  remains  constant  for  a  certain  period 
until  it  finally  disappears.  If  at  a  time  when  the  serum  contains  a  certain 
amount  of  antitoxin  a  new  inoculation  is  undertaken,  the  so-called  "  negative 
phase,"  sets  in,  i.e.,  the  amount  of  antitoxin  within  the  serum  first  sinks  and 
then  is  followed  by  a  compensatory  rise.  By  becoming  acquainted  with  the 
wave-like  fluctuations  in  the  antitoxin  content  of  the  serum,  and  renewing 
the  injection  at  the  time  of  highest  content,  one  can  produce  a  serum  with 


DIPHTHERIA    SERUM.  73 

very  strong  antitoxic  qualities.  This  was  done  by  Salomonsen  and  Madsen 
who  by  experimentation  found  that  the  maximum  height  of  the  antitoxic 
curve  was  reached  on  the  tenth  day  after  each  inoculation.  For  this  reason 
it  is  wise  to  choose  this  day  for  the  removal  of  the  serum.  As  regards  other 
sera,  e.g.,  tetanus,  different  periods  have  been  empirically  found  to  be  most 
serviceable.  As  the  antitoxic  curve  does  not  remain  at  a  high  point  for  a 
long  time,  the  injections  should  be  repeated  from  time  to  time.  For 
highly  immunized  horses,  monthly  injections  usually  suffice. 

After  the  serum  has  been  obtained,  the  important  problem  which  arises 
is  how  to  keep  it  sterile.  This  is  accomplished  by  aseptic  precautions  at  the 
time  of  the  obtention  of  the  serum  and  eventually  by  the  addition  of  pre- 
servatives such  as  1/2  per  cent,  carbolic. 

This  procedure  finished,  the  next  step  is  to  estimate  the  amount  of  the 
antitoxin  content  in  the  serum. 

According  to  v.  Behring  and  Boer,  the  value  of  the  serum  should  be 
ascertained  in  respect  to  its: 

i.  Protective  power 


,  against  infection. 

2.  Curative  power     J 

3.  Protective  power   1 

~        .  >  against  intoxication. 

4.  Curative  power     J 

v.  Behring  found  that  these  four  properties  run  parallel  with  each  other 

so  that  for  practical  purposes,  it  suffices  to  establish  only  one  of  these  qualities. 

For    diphtheria   serum  it    has   proved  most  serviceable   to 

Standardiza-  estimate  the   strength  of  the  immunity  against  intoxication, 

.      since  one  is  dealing  with  a  purely  antitoxic  serum. 
Diphtheria 

Serum.        Behring's  original  mode  of  standardization  consisted  in  gradually  add- 
ing doses  of  serum  to  the  minimal  lethal  dose  of  toxin  and  injecting  the 
mixtures  into  guinea-pigs,  thus  determining  the  smallest  amount  of  serum  capable  of 
preventing  death  of  the  animal.     It  was  soon  found,  however,  that  this  method  gave 
too  inconstant  results  because  the  individual  minimal  lethal  dose  was  too  variable. 

Ehrlich,  therefore,  modified  the  process  by  using  ten  times  the  minimum  lethal  dose. 
This  amount  of  toxin,  mixed  with  decreasing  amounts  of  serum  and  made  up  to  4  c.c. 
with  physiological  salt  solution  was  injected  subcutaneously  into  a  guinea-pig.  The 
smallest  amount  of  serum  which  saved  it  from  being  killed  on  the  fourth  to  fifth  day  was 
thus  estimated. 

The  method  of  standardization  used  at  the  present  time  owes  its  origin 
to  Ehrlich. 

In  order  to  attain  uniformity  in  the  comparative  value  of  all  sera, 
Behring  and  Ehrlich  recommended  the  adoption  of  two  empirical  values; 
"the  normal  toxin,"  and  the  "normal  curative  serum." 

The  normal  diphtheria  toxin  is  one  which  contains  enough  toxin  in  i  c.c. 
to  kill  25,000  gm.  of  guinea-pigs  or  100  guinea-pigs  each  weighing  250  gm. 

A  normal  curative  serum  is  one  of  which  o.i  c.c.  suffices  to  neutralize 


74  TOXIN  AND   ANTITOXIN. 

i  c.c.  of  Behring's  normal  poison,  i.e.,  is  able  to  overcome  the  effect  of  100 
fatal  doses,  i  c.c.  of  this  normal  curative  serum  represents  one  immunity 
or  antitoxin  unit. 

The  present  antitoxin  unit  was  fixed  by  Ehrlich  who  adopted  that 
amount  of  antitoxin  as  his  standard,  which  when  mixed  with  100  times  the 
lethal  dose  of  a  then  existing  toxin,  and  injected  into  an  animal,  was  sufficient 
to  so  neutralize  the  toxin  that  not  the  slightest  evidence  of  either  a  local 
symptom  or  general  illness  was  present.  Ehrlich  chose  the  antitoxin  rather 
than  the  toxin  as  the  constant  of  standardization,  because  the  toxin  would 
deteriorate  after  some  time,  while  the  antitoxin  could  be  preserved  in  a 
stable,  unchangeable  form. 

In  spite  of  this  fact,  the  new  method  of  titration  was  still  unsatisfactory, 
inasmuch  as  the  toxin  could  undergo  other  biological  changes  not  yet  taken 
into  account. — To  understand  these,  the  acquaintance  of  several  new  terms 
is  essential,  and  they  are,  dosis  eerie  efficax,  limes  +  or  limes  death,  limes  o 
or  limes  zero. 

While  the  dosis  letalis  minima  represents  the  smallest  dose  of  toxin 
which  may  be  fatal  in  four  to  five  days,  the  dosis  certe  efficax  (dose  of  certain 
efficiency)  stands  for  the  smallest  dose  which  will  surely  kill  any  pig  of  250 
gm.  within  this  period  of  time. 

By  limes  +  (limes  death)  is  meant  the  smallest  amount  of  toxin  which 
after  being  mixed  with  an  antitoxin  unit,  will  still  cause  the  death  of  a 
guinea-pig  within  four  to  five  days.  By  limes  o  (limes  zero)  is  understood 
the  dose  of  toxin  which  is  just  neutralized  by  one  antitoxin  unit  (I.  E.  = 
antitoxin  unit  or  ."Immunitats  Einheit"),  so  that  no  toxin  is  free  and  the 
animal  remains  perfectly  well.  Limes  +  therefore  implies  an  excess  of 
poisonous  toxin;  L  O,  perfect  neutralization. 

Theoretically  speaking,  the  difference  between  L  +  and  L  O  should 
represent  the  minimum  lethal  dose  (d.  1.  m.). .  This,  however,  is  almost  never 
so,  as  is  shown  in  the  following  illustration. 

The  d.  1.  m.  of  a  certain  poison  was  estimated  as  0.0039  c.c. 
L+  was  found  to  be  0.48  c.c.  =123  lethal  doses. 
LO  was  found  to  be  0.42  c.c.  =108  lethal  doses. 


Difference  0.06  cm.  =   15  lethal  doses. 

In  order  to  explain  this  phenomenon  Ehrlich  considered  that  there  were 
two  other  substances  contained  within  the  diphtheria  bouillon  in  addition  to 
the  diphtheria   toxin;   namely,   diphtheria  toxon  and  diphtheria  toxoid. 
The  toxon  is  a  poison  which  in  contrast  to  the  toxin  has  only  a 
Toxon.      slight  affinity  for  the  antitoxin.     It  is  this  body  which  is  prob- 
ably the  cause  of  the  paralysis  occurring  weeks  after  the  infection. 
In  a  mixture  like  L  O,  the  antitoxin  has  fully  neutralized  both  the  toxon 
as  well  as  the  toxin.     If,  however,  more  diphtheria  poison  is  added  to  the 


TOXOID. 


75 


I.  E.,  as  is  done  in  the  L  + ,  the  antitoxin,  on  account  of  its  greater  attrac- 
tion for  the  toxin,  will  combine  with  the  latter  and  leave  the  toxon  free  to 
subsequently  carry  out  its  own  functions.  The  more  crude  poison  is  added, 
the  more  toxon  remains  unbound,  until  a  point  is  reached  when  no  more 
toxin  can  be  taken  up  and  consequently  some  is  left  unneutralized.  If  the 
amount  of  active  toxin  reaches  the  dosis  letalis  minima,  it  is  sufficient  to 
kill  the  animal  and  thus  the  limes  -f  is  attained. 

When,  instead  of  freshly  prepared  toxins  Ehrlich  employed  older  bouillon 
cultures,  the  poisonous  qualities  distinctly  sank  to  about  one-half,  but  the 
surprising  fact  was  that  the  L  +  had  not  been  altered  and  even  though  it 
had  lost  one-half  of  its  toxic  power,  it  had  still  retained  its  initial  potential- 
ities for  neutralizing  antitoxin. 

Ehrlich's  explanation  was  that  the  diphtheria  poison  consists  of  two 
Toxoid.        molecular  groups;  one  the  carrier  of  the  toxic  qualities,  and  therefore 
known  as  the  "toxophore"  group,  the  other  uniting  with  the  antitoxin 
and  having  the  capability  of  neutralizing  it,  known  as  the  "haptophore  group."     The 
toxophore  group  is  very  labile,  while  the  haptophore  group  strongly  in  contrast  to  it,  is  char- 
acterized by  its  stability.     The  toxophore  element  destroyed,  the  diphtheria  poison  loses 
its  toxic  qualities,  but  retains  its  power  to  bind  antitoxin.     A  non-poisonous  diphtheria 
toxin  possessing  such  power,  is  designated  by  Ehrlich  as  " Diphtheria  Toxoid." 

The  mode  of  standardization  of  serum  advocated  at  the  present  day  is  ap- 
plicable exclusively  to  the  L  -f-  dose.  It  is  effected  by  injecting  guinea-pigs 
subcutaneously  with  mixtures  of  various  doses  of  diphtheria  toxin  on  hand, 
plus  an  anti-toxin  unit,  and  noting  the  smallest  amount  of  toxin  which  kills  the 
animal  in  four  to  five  days.  This  L  -f-  as  the  constant  factor  is  now  mixed 
with  different  amounts  of  the  serum  to  be  tested  and  that  quantity  deter- 
mined which  just  prevents  the  death  of  the  animal.  If  for  example  i/ioo 
c.c.  is  necessary,  this  serum  is  considered  one  hundred  times  as  strong 
as  the  standard  antitoxin  unit,  or  in  other  words  it  contains  100  immunity 
units. 

This  method  of  Ehrlich  has  been  adopted  not  only  in  Germany,  but  almost  in  all 
other  countries  in  Europe  and  also  in  America.  Only  in  France  the  principle  varies 
somewhat,  as  here  the  serum  is  tested  both  for  its  protective  and  curative  action.  The 
protective  power  of  a  serum  is  considered  50,000  if  o.oi  c.c.  of  a  serum  saves  a  guinea- 
pig  weighing  500  gm.  from  the  fatal  consequences  following  a  dose  of  toxin  sufficient  to 
kill  an  animal  of  the  same  weight  in  thirty  to  forty  hours.  The  standard  therefore 
takes  into  consideration  the  relation  between  the  amount  of  serum  and  the  weight  of 
the  animal.  The  serum  is  injected  into  the  guinea-pig  twelve  hours  before  the  toxin  and 
the  animal  should  not  lose  in  weight  during  the  following  six  days.  The  curative 
power  is  estimated  by  injecting  a  guinea-pig  with  a  dose  of  toxin  (sufficient  to  kill  a 
control  animal  in  thirty  to  forty  hours)  and  six  hours  afterward  the  serum  is  injected. 
The  animals  remaining  alive  on  the  sixth  day  are  considered  as  cured. 

The  French  method  of  standardization  is  built  upon  the  belief  of  Roux 
that  no  parallelism  necessarily  exists  between  the  protective  and  curative 
values  of  a  serum.  Kraus  and  Schwarz  have  recently  published  accounts 


76 


TOXIN  AND  ANTITOXIN. 


of  experiments  which  corroborate  Roux's  views.  They  claim  that  a  very 
highly  valent  diphtheria  serum  has  a  lower  curative  value  than  one  less  so; 
that  the  curative  power  of  a  serum  does  not  depend  upon  the  increase  or 
decrease  of  the  antitoxin  content  during  the  immunization  of  an  animal, 
and  that  Ehrlich's  process  of  standardization  taking  into  consideration  only 
the  protective  power,  requires  additional  modification.  Berghaus  working 
in  Ehrlich's  Institute  answered  the  above  exceptions  in  so  satisfactory  a 
manner,  that  up  to  the  present  day  Ehrlich's  views  are  still  upheld  by  the 
majority  of  workers  in  this  field. 

While  in  some  countries  the  government  institutes  have  complete  control  over  the 
production  of  diphtheria  serum,  in  Germany  it  is  manufactured  by  private  concerns,  but 
under  government  supervision. 

The  serum  must  be  absolutely  clear,  free  of  bacteria  and  toxins,  especially  tetanus 
toxin,  and  must  not  contain  more  than  1/2  per  cent,  of  phenol.  It  should  contain  at 
least  the  number  of  antitoxin  units  designated  by  the  factory. 

In  the  United  States  the  standard  antitoxin  is  distributed  by  the  Public  Health  and 
Marine  Hospital  Service  Laboratories.  Since  1902  the  production  and  sale  of  diphtheria 
antitoxin  has  been  regulated  by  law. 

At  frequent  intervals,  antitoxin  is  bought  in  the  open  market  and  examined  at  the 
hygienic  laboratories  of  the  United  States  Public  Health  and  Marine  Hospital  Service. 
Antitoxic  serum  containing  less  than  a  hundred  units  to  each  cubic  centimeter  is  pre- 
cluded from  sale. 

The  Serum  Therapy  of  Diphtheria. 

In  man  the  antitoxic  diphtheria  serum  is  used  with  success  for  both 
curative  and  prophylactic  purposes. 

For  therapeutic  application  it  is  of  the  greatest  importance  to  employ  the 
serum  in  sufficient  quantities  and  as  soon  as  possible.  The  value  of  early 
intervention  can  be  seen  from  the  following  chart  of  Kossel : 


Day  of  illness. 

Treated. 

Cured. 

Died. 

Percentage  of  cures. 

i 

7 

7 

o 

IOO 

2 

7i 

69 

2 

97 

3 

30 

26 

4 

87 

4 

39 

30 

9 

77 

5 

25 

15 

10 

60 

6 

17 

9 

8 

47 

7-14 

4i 

21 

20 

5i 

Indefinite 

2 

j 

233 

179 

54 

77 

Large  doses  of  antitoxin  should  be  administered  right  from  the  start.     The 
old  practice,  still  employed  by  few,  of  using  small  doses  is  to  be  condemned, 


SERUM    THERAPY    OF    DIPHTHERIA. 


77 


for  the  aim  in  the  treatment  is  to  neutralize  as  soon  as  possible  all  the  free 
and  partly  bound  toxin. 

According  to  the  researches  of  Doenitz,  more  recently  confirmed  and 
extended  by  Fritz  Meyer,  it  was  established  that  large  amounts  of  antitoxin 
can  even  neutralize  toxin  already  attached  to  the  tissue  cells.  Men  with 
practical  experience  like  Heubner,  give  4000  units  as  the  initial  dose.  In 
the  United  States  doses  as  high  as  10,000  to  100,000  I.  E.  have  been  admin- 
istered with  good  results.  The  view  of  large  dosage  is  being  gradually  taken 
up  also  in  Germany.  At  any  rate  it  is  by  far  better  to  give  too  much  than  too 
little.  If  the  first  injection  does  not  suffice  it  should  be  repeated  the  next  day. 
The  only  possible  drawback  associated  with  the  use  of  excessive  amounts  is 
the  possibility  of  serum  sickness,  to  be  mentioned  later.  Netter  has  found 
that  the  administration  of  i  gm.  of  calcium  chloride  on  three  successive  days 
prevents  serum  sickness. 

The  serum  has  thus  far  been  as  a  rule  injected  subcutaneously.  This 
method  is  very  practical  and  as  far  as  anaphylaxis  is  concerned,  is  the  least 
dangerous.  The  disadvantage,  however,  is  that  it  is  very  slowly  absorbed. 
Madsen  and  Hendersen-Smith  have  shown  that  but  a  trace  of  antitoxin  can 
be  found  in  the  blood  of  the  patient  four  and  three-fourth  hours  after  the 
injection,  and  only  after  two  to  three  days  can  larger  amounts  be  demon- 
strated. In  view  of  this,  Morgenroth  recommends  the  gluteal  intramuscular 
injection  for  here  a  much  more  rapid  absorption  follows.  In  cases  of  danger- 
ous illness  intravenous  injection  may  be  undertaken.  For  this  purpose 
Meyer  advises  a  serum  free  of  carbolic  acid,  although  this  is  not  absolutely 
necessary. 

The  importance  of  the  method  of  injection  is  clearly  shown  by  the  comparative 
experiments  of  Berghaus.     In  order  to  save  a  guinea-pig  injected  with  a  definite  amount 
of  toxin  and  followed  in  i  hour  by  antitoxin,  it  was  necessary  to  employ: 
0.08  I.  E.  by  intracardial  injection. 
7.0    I.E.  by  intraperitoneal  injection. 
40.00  I.  E.  by  subcutaneous  injection. 

Thus  the  curative  power  was  increased  500  fold  by  placing  the  antitoxin  directly  into  the 
circulation. 

The  treatment  of  diphtheria  must  by  no  means  be  limited  to  serum 
therapy.  A  symptom  of  grave  prognosis  is  the  lowered  blood  pressure 
which  must  be  counteracted  by  infusions  of  1/2  liter  of  physiological  salt 
solution  containing  five  to  six  drops  of  adrenalin. 

The  question  whether  the  use  of  concentrated  antitoxin  is  therapeutically 
more  efficient  than  the  non-concentrated  is  still  a  matter  for  discussion. 
Numerous  authors  claim  that  sera  of  medium  strengths  (about  400  I.  E.) 
are  most  efficient.  The  highly  concentrated  sera  are  much  more  expensive. 

For  prophylactic  purposes  500  to  1,000  units  injected  subcutaneously 
usually  suffice.  Protection  thus  attained  lasts  about  three  weeks. 


CHAPTER  VIII. 
TOXIN  AND  ANTITOXIN  (continued). 

DEFINITION  OF  TOXIN,  TETANUS  TOXIN,  BOTULISM  TOXIN,  DYSENTERY  TOXIN, 

STAPHYLOLYSIN. 

The  diphtheria  toxin  and  its  antitoxin  just  discussed  in  detail  is  of  great 
practical  and  theoretical  importance,  and  can  serve  as  a  type  of  all  true  toxins 
and  antitoxins.  Bacterial  toxins  can  be  defined  as  poisons  given  off  by  the  bac- 
teria, the  symptoms  resulting  from  their  action  appearing  after  a  certain  incuba- 
tion period.  The  invaded  organism  reacts  by  the  production  of  specific  anti- 
toxins which  neutralize  the  toxins  in  amounts,  following  the  law  of  multiple 
proportions. 

Further  analysis  of  this  definition  indicates  that  a  substance  can  be  con- 
sidered a  toxin  only  when  it  has  a  poisonous  action,  or  in  the  words  of 
Ehrlich  when  it  possesses  a  toxophore  group . 

This  toxicity  does  not  always  manifest  itself  by  necrosis  or  death  as  in 
diphtheria.  More  frequently  the  toxin  has  a  somewhat  selective  action 
affecting  a  certain  group  of  organs.  Thus  a  toxin  acting  upon  the  central 
nervous  system  or  blood  is  designated  respectively  as  a  neurotoxin  or  a 
hemotoxin.  To  differentiate  a  true  toxin  from  other  poisonous  products 
obtained  from  bacteria,  it  is  important  to  note  that  all  true  toxins  are 
elements  of  secretion  of  the  living  bacteria,  and  can  be  separated  from  them 
by  filtration.  According  to  this  definition  poisons  contained  within  the  bac- 
terial bodies  themselves,  which  may  be  liberated  by  various  mechanical, 
physical,  or  chemical  means,  cannot  be  considered  as  belonging  to  the 
class  of  true  toxins.  These  poisons  are  characterized  by  peculiar  proper- 
ties and  are  known  as  endotoxins.  In  addition  it  may  be  remarked  that 
inasmuch  as  a  true  toxin  requires,  a  period  of  incubation  in  order  to  mani- 
fest its  action,  those  toxins  which  act  spontaneously  are  to  be  excluded  from 
the  former  group.  R.  Krause  nevertheless  considered  some  of  the  poisons 
isolated  from  the  cholera  and  cholera-like  spirilla  (El  Tor  Vibrio)  as  true 
toxins  even  though  they  lack  an  incubation  period. 

The  real  essential  property  of  a  toxin  is  doubtlessly  that  one  can  immu- 
nize against  it,  and  be  able  to  demonstrate  the  presence  of  antitoxins  within 
the  serum  of  the  immunized  animal.  Ehrlich  furthermore  claims  that  the 
amount  of  antitoxin  produced  follows  the  law  of  multiple  proportions.  By 

78 


TETANUS    TOXIN.  79 

this  is  meant  that  the  relationship  between  a  definite  dose  of  toxin  and  the 
amount  of  antitoxin  just  sufficient  to  neutralize  it  is  constant,  so  that  if  ten 
volumes  of  toxin  hold  in  bounds  ten  volumes  of  antitoxin,  100  volumes  of 
toxin  neutralize  100  volumes  of  antitoxin.  This  relation  is  best  exemplified 
by  the  diphtheria  toxin  and  antitoxin.  With  the  other  toxins,  conditions  are 
more  complicated  so  that  many  objections  have  been  raised  against  the 
above  rule  of  multiple  proportions.  (Bordet,  Arrhenius,  Madsen,  etc.) 
The  true  toxins  causing  infections  in  man  are  limited  to  the 

1.  Diphtheria  toxin. 

2.  Tetanus  toxin. 

3.  Botulism  toxin. 

4.  Dysentery  toxin. 

5.  Staphylolysin  and  similar  bacterial  hemotoxins. 

Tetanus  Toxin. 

The  tetanus  toxin  is  found  within  filtrates  of  bouillon  cultures 
Tetanus  of  the  tetanus  bacillus.  While  partial  erobiosis  does  not 
Toxin.  entirely  eliminate  toxin  formation,  anerobic  conditions  are  by 

far  more  favorable  for  it.  The  tetanus  toxin  is  of  two  kinds; 
the  tetanospasmin,  and  tetanolysin;  the  former  a  neuro toxin,  the  latter  a 
hemotoxin.  The  tetanospasmin  is  the  more  important  of  the  two  for  the 
reason  that  it  is  the  agent  which  produces  convulsions.  If  susceptible 
animals  such  as  mice  or  guinea-pigs  are  injected  subcutaneously  or  intra- 
muscularly with,  tetanus  toxin,  after  a  certain  interval — the  incubation 
period — they  will  begin  to  show  symptoms  due  to  tetanospasmin.  They 
become  hypersensitive  to  reflex  stimulation;  clonic  convulsions  and  toxic 
rigidity  of  the  muscles  set  in.  In  animals  the  last  state  appears  first  in  the 
group  of  muscles  nearest  the  point  of  injection,  while  in  man  the  spasm 
almost  regularly  starts  in  the  muscles  of  the  lower  jaw.  By  intravenous 
and  intraperitoneal  injections,  the  tetanic  spasm  appears  simultaneously  in  all 
muscles  of  the  body;  on  intracerebral  inoculation,  Roux  and  Borrel  describe 
the  occurrence  of  epileptiform  seizures,  polyuria  and  certain  motor  disturb- 
ances— the  entire  set  of  complications  being  known  as  cerebral  tetanus. 
Rabbits  receiving  very  small  amounts  of  tetanus  toxin  intravenously  die  after 
gradual  emaciation  and  marked  cachexia.  This  type  of  infection  is  desig- 
nated by  Doenitz  as  tetanus  sine  tetano.  If  taken  per  os,  tetanus  toxin 
manifests  no  poisonous  symptoms.  Tetanospasmin  is  a  distinct  nerve  poison 
especially  affecting  the  central  nervous  system. 

Experiments  by  Wassermann  and  Takaki  have  demonstrated  an  especially  close 
affinity  existing  between  the  tetanus  toxin  and  certain  organs.  These  organs  differ  in 
different  animals.  Thus  in  man,  horse,  and  guinea-pig  only  the  central  nervous  system, 
while  in  rabbits  in  addition  to  this,  also  the  liver  and  spleen  take  up  the  tetanus  poison. 


8o  TOXIN  AND   ANTITOXIN. 

If  an  emulsion  of  brain  tissue  and  a  fatal  dose  of  tetanus  toxin  are  mixed  and  the  mixture 
injected  into  mice,  the  latter  remain  unaffected.  According  to  Doenitz  only  the  gray 
matter  and  not  the  white  substance  of  the  brain  possesses  this  absorption  power.  If  the 
brain  emulsion  is  boiled,  however,  it  loses  this  affinity  for  the  toxin. 

Concerning  the  way  by  which  the  toxin  reaches  the  central  nervous  sys- 
tem, opinions  vary.  Most  writers,  especially  Meyer  and  Ransom,  consider 
that  the  journey  is  made  along  the  nerve  paths.  Zupnik  on  the  other  hand 
believes  that  it  is  distributed  through  the  blood  stream  and  is  taken  up  not 
only  by  the  nervous  system,  but  also  to  a  great  extent  by  the  muscles. 

That  tetanus  toxin  is  very  labile  is  well  known.  According  to  Kitasato, 
five  minutes  at  65°  C.  or  twenty  minutes  at  60°  is  sufficient  to  weaken  the 
toxicity  to  a  great  extent,  in  fact  even  almost  to  destroy  it.  Light  has  a 
similar  effect  upon  it.  Careful  as  its  preservation  may  be,  the  soluble  tetanus 
toxin  soon  becomes  attenuated.  Hence  the  best  way  of  keeping  it  in  stock  is 
in  a  dry  form.  For  estimating  the  strength  of  the  toxin  white  mice  are  em- 
ployed and  are  subcutaneously  injected  with  fresh  soluble  toxin,  the  lethal 
dose  being  the  amount  which  kills  the  animals  in  four  to  five  days.  Animals 
more  susceptible  than  mice  are  horses,  they  being  twelve  times  as  sensitive 
and  guinea-pigs  six  times  as  much.  Hens  possess  greater  power  of  resist- 
ance, being  30,000  times  less  susceptible  to  the  toxin  than  mice. 

Tetanolysin  acts  upon  the  red  blood  cells  and  disintegrates  them.  The 
erythrocytes  of  goats,  sheep  and  horses,  are  best  suited  for  experiments  to 
demonstrate  this  action.  Ehrlich  showed  that  the  tetanolysin  and  the 
tetanospasmin  are  really  two  identically  different  toxins  and  not  one  toxin 
with  a  twofold  function.  When  tetanus  poison  is  mixed  with  red  blood 
cells  the  tetanolysin  is  absorbed  and  the  tetanospasmin  remains  free.  Even 
the  antitoxins  of  these  two  are  different. 

As  far  as  the  standardization  of  the  tetanus  serum  is  concerned,  it  is 
affected  on  the  same  lines  as  the  diphtheria  serum,  i.e.,  the  L  +  dose  of  toxin 
being  the  one  employed. 

"In  America  the  method  of  standardization  was  regulated  by  a  law  passed 
in  July,  1908,  based  upon  the  work  of  Rosenau  and  Anderson  at  the  United 
States  Hygienic  Laboratories  at  Washington.  Their  unit  of  antitoxin  is  ten 
times  the  smallest  amount  of  serum  necessary  to  save  the  life  of  a  guinea-pig 
for  ninety-six  hours,  against  the  official  unit  of  standard  toxin.  This  toxin 
unit  consists  of  100  minimal  lethal  doses  of  a  precipitated  toxin  preserved  at 
the  hygienic  laboratory  of  the  Public  Health  and  Marine  Hospital  Service. 
At  the  hygienic  laboratory  at  Washington  a  standard  toxin  and  antitoxin 
are  preserved  under  special  conditions,  and  standard  toxin  and  antitoxin, 
arbitrary  in  their  first  establishment,  are  kept  constant  by  being  meas- 
ured against  each  other  from  time  to  time.  For  details  of  this  standardi- 
zation the  original  article  in  the  United  States  Hygienic  Laboratory  Bulletin 
43,  1903,  should  be  consulted." 


BOTULISM   TOXIN.  8 1 

In  regards   to  the   efficiency  of  serum  therapy  in  tetanus, 

TherT^of     °Pinions  differ-     There  is,  however,  no  doubt  that  a  certain 

Tetanus.      amount  of  reliance  can  be  placed  upon  this  treatment.     Failures 

in  successful  application  are  ascribed  to  the  different  paths  by 
which  the  toxin  and  antitoxin  travel.  The  former  is  carried  along  by  the 
nerve  fibers,  while  the  latter  by  the  blood  stream.  Thus  the  serum  instead 
of  being  given  subcutaneously,  as  is  the  general  rule,  is  administered  by 
intraneural,  intracerebral,  and  subdural  injections.  100  to  200  units 
should  be  injected  subcutaneously  at  the  site  of  the  infection  or  its  vicinity 
and  in  addition  the  nerve  fibers  supplying  the  infected  region  should  be 
exposed  and  inoculated  with  moderate  doses  of  antitoxin  at  various  points 
along  their  centripetal  course. 

The  prophylactic  use  of  tetanus  serum  has  met  with  better  results. 
Behring  advises  the  administration  of  ten  to  twenty  antitoxin  units  subcutane- 
ously. Calmette  sprinkles  upon  the  open  navel  at  birth  a  powder  made  of 
dried  serum  as  a  prophylactic  against  tetanus  neonatorum.  Bocken- 
heimer  advises  an  ointment  containing  the  antitoxin  as  a  dressing  for  sus- 
picious wounds. 

The  Botulism  toxin  is  the  poison   produced  by  the  bacillus 

Botulism      botulinus.     This  is  the  exciting  agent  of  a  type  of  meat  and 

Toxin.       sausage  poisoning  described  by  van  Ermenghem  in  1896  as 

Botulism.  The  bacillus  botulinus  is  a  very  actively  motile 
anerobic  bacterium  which  grows  at  room  temperature  and  presents 
marked  gas  and  toxin  formation.  A  medium  in  which  the  toxin  is  readily 
produced  consists,  according  to  Ermenghem,  of  an  alkaline  bouillon  made 
in  the  form  of  an  infusion  from  ham  with  the  addition  of  i  per  cent,  of  glu- 
cose, i  per  cent,  of  peptone  and  i  per  cent,  of  NaCl. 

The  toxin  can  thus  be  demonstrated  after  3  weeks  of  growth,  and  is  then 
obtained  by  bacterial  filtration.  The  cultures  have  a  sour  odor  like  unto 
butyric  a  :id.  The  toxin  deteriorates  easily  when  exposed  to  air  and  light. 
It  is  therefore  preserved  in  brown,  sealed  vials,  and  kept  on  ice;  or,  in  a 
dried  form  in  vacuum.  Heating  the  toxin  for  three  hours  at  58°  or  one-half 
hour  at  80°  destroys  its  toxicity. 

Acting  unrestrained,  the  botulism  toxin  is  one  of  the  severest  of  poisons. 
It  affects  susceptible  animals  even  in  minutest  doses.  In  contradistinction 
to  other  toxins  it  is  fatal  even  when  taken  per  os. 

The  characteristic  symptoms  produced  by  botulism  intoxication  consist  of  hyper- 
secretion  of  mucus  from  the  mouth  and  nose,  paralysis  of  eye  muscles,  urine  retention, 
obstipation,  dysphagia,  aphagia,  and  aphoria.  No  fever,  nor  any  sensatory  disturbances 
are  in  evidence.  Death  takes  place  because  of  bulbar  paralysis  accompanied  by  re- 
spiratory and  cardiac  failure. 

The  poison  is  absorbed  or  arrested  in  the  central  nervous  system,     o.i 
c.c.  of  an  emulsion  of  central  nervous  tissue  neutralizes  three  times  the  fatal 
6 


82 


TOXIN  AND  ANTITOXIN. 


dose  for  mice.     Lecithin,  cholesterin,  as  well  as  fatty  substances  like  butter 
and  oil,  act  in  a  similar  manner. 

Monkeys,  rabbits,  guinea-pigs,  mice  and  cats  are  susceptible  to  the  toxin. 

Cats  usually  exhibit  the  most  characteristic  clinical  picture.  Localized  and  almost 
pathognomonic  paralyses  occur  in  the  form  of  prolapse  of  tongue,  marked  mydriasis, 
aphonia,  aphagia,  etc. 

In  mice,  paralysis  of  the  hind  extremities  sets  in  after  quite  a  small  dose;  and  death 
follows  in  a  few  hours. 

In  rabbits  and  guinea-pigs,  moderate  doses  (0.0003-0.001  c.c.)  occasion  no  manifesta- 
tions during  the  first  two  to  three  days,  but  subsequently,  the  above  mentioned  paralyses 
arise  and  several  hours  after  the  animals  expire.  With  larger  doses  (o.i  to  0.5  c.c.)  the 
incubation  period  lasts  only  a  couple  of  hours  and  then  dyspneic  attacks  usually  succeeded 
by  motor  paralysis  and  death  are  the  consequences. 

The  strength  of  the  botulism  toxin  is  ascertained  by  injecting  guinea-pigs  subcutane- 
ously  and  observing  the  time  when  loss  in  weight,  flabbiness  of  abdominal  muscles  and 
death  occur. 

The  following  chart  by  Madsen  exhibits  the  above  principle. 


Dose  in  c.c. 

Result. 

Dose  in  c.c. 

Result. 

0.0015 

+  on  i  st.  day 

o  .  0009 

Weakness  in  3  weeks. 

0.0015 

+  after  i  1/2  days 

o  .  0009 

Loss  in  weight. 

0.0015 

+  after  2         days 

0.0009 

Loss  in  weight. 

0.0013 

+  after  2         days 

0.0007 

Loss  in  weight  in  2  weeks. 

0.0013 

+  after  5         days 

o  .  0007 

Loss  in  weight  in  i  week. 

0.0013 

+  after  6         days 

o  .  0007 

Loss  in  weight  in  i  week. 

O.OOI 

+  after  4         days 

o  .  0005 

Loss  in  weight  in  i  week. 

0.001 

+  after  5         days 

o  .  0003 

Practically  no  symptoms;  only 

O.OOI 

+  after  51/2  days 

several  days  of  weakness. 

Kempner  immunized  goats  against  Botulism  toxin  and  proved  the  presence  of  anti- 
toxins within  their  sera.  Immunization  of  rabbits  and  guinea-pigs  is  only  feasible  if 
primary  inoculations  are  made  with  a  toxin  previously  attenuated  by  heat  for  one-half 
hour  at  60°  C. 

Recently  Wassermann  has  immunized  horses  against  this  toxin.  In 
mew  of  the  high  mortality  and  lack  of  any  other  specific  medication,  the  use  of 
this  serum  is  strongly  advised.  In  animal  experimentation  it  shows  itself 
of  undeniable  value.  As  for  its  effects  in  man,  it  has  not  been  employed 
frequently  enough  to  judge. 

The  botulism  toxin  and  antitoxin  unite  only  very  slowly.  Otto  and  Sachs  have  shown 
that  the  inoculation  of  rabbits  with  a  three  hours  old  mixture  of  toxin  and  antitoxin 
occasioned  greater  toxic  effects  when  administered  intravenously  than  when  given 
subcutaneously.  Only  in  mixtures  twenty-four  hours  old  was  this  difference  overcome. 


DYSENTERY   TOXIN.  83 

Dysentery  toxin  was  first  demonstrated  by  Conradi.     Subse- 

Dysentery     quently  from  experiments  by  Rosenthal,  Todd,  Kraus  and 

Toxin.        Doerr,  etc.,  it  became  evident  that  this  was  a  true  toxin  and  not 

an  endotoxin  as  was  originally  considered.  Only  the  Kruse- 
Shiga  type  of  bacillus  forms  a  toxin;  for  the  Flexner  kind,  no  definite  toxin  has 
as  yet  been  isolated.  Recent  investigators,  however,  especially  Kraus  and 
Doerr  are  inclined  to  consider  the  human  dysentery  of  the  Kruse-Shiga 
origin  in  the  light  of  an  intoxication  or  toxemia  similar  to  diphtheria.  The 
lesions  in  the  large  intestine  where  the  bacteria  accumulate  can  be  compared 
to  the  diseased  diphtheria  tonsils,  while  the  other  manifestations,  as  the 
central  symptoms,  cardiac  disturbances,  nervous  sequelae,  eye  affections, 
etc.,  can  be  taken  as  expressions  of  the  toxemia. 

Like  the  other  described  toxins,  the  dysentery  toxin  can  be  obtained  by  filtration  of 
bouillon  cultures.  The  meat  infusion  must  be  quite  alkaline.  The  optimum  alkalinity, 
according  to  Doerr,  is  obtained  by  adding  0.3  per  cent,  soda  to  litmus  neutral  bouillon. 
The  precipitate  thus  formed  which  increases  on  sterilization  should  not  be  removed  by 
filtration.  Doerr  also  advises  finely  powdered  chalk  (20  gm.  pro  liter)  to  be  added  to  the 
weakly  alkaline  bouillon  before  the  last  sterilization.  The  toxin  is  formed  very  gradually; 
the  maximum  is  derived  after  two  to  three  weeks.  The  gray  white  pellicle  upon  the  sur- 
face of  the  culture  can  be  taken  as  an  indicator  for  the  amount  of  toxin  present. 

According  to  Kraus  a  good  dysentery  toxin  can  also  be  made  by  emulsifying  the 
bacteria  (grown  upon  agar)  in  physiological  salt  solution  and  filtering  this  through  Reichel 
filters. 

The  toxicity  of  individual  strains  of  dysentery  bacilli  varies  greatly. 

The  strength  of  the  toxin  is  diminished  by  heating  for  one  to  two  hours  at  60°  C. 
Higher  temperatures  destroy  it,  as  80°  C.  where  destruction  occurs  in  three  minutes  and 
90°  to  100°  C.  in  one  minute. 

Acids  destroy  the  toxin  probably  by  the  formation  of  a  non-poisonous  compound. 
The  addition  of  a  strong  alkali  restores  the  toxicity. 

Its  preservation  can  be  accomplished  in  a  fluid  state  under  the  cover  of  toluol. 

The  action  of  dysentery  toxin  can  best  be  studied  by  its  effect  upon  rab- 
bits after  intravenous  inoculation.  Large  doses  kill  the  animals  in  very 
short  time,  six  to  seven  hours.  The  ordinary  lethal  dose  produces  charac- 
teristic symptoms  consisting  of  paresis,  diarrhea,  which  may  be  bloody, 
paralysis  of  the  bladder,  hypothermia,  etc.  Death  takes  place  in  three  to 
four  weeks. 

Given  subcutaneously,  or  intraperitoneally,  the  toxin  has  only  a  very 
mild  action.  The  incubation  period  is  especially  prolonged.  Given  per  os, 
no  effect  is  in  evidence. 

Besides  rabbits  the  other  susceptible  animals  are  monkeys,  cats  and  dogs  (to  large 
doses) ;  chickens,  pigeons  and  guinea-pigs  are,  in  the  opinion  of  Kraus  and  Doerr,  not  at 
all  affected  by  the  toxin. 

The  intestinal  changes  found  at  post-mortem  examination  of  the  animals  very  closely 
simulate  the  pathological  alterations  occurring  in  man.  A  hemorrhagic  necrotic  enteritis 
is  present  which  in  rabbits  is  regularly  localized  in  the  appendix  and  cecum,  while  in  dogs 


84  TOXIN  AND  ANTITOXIN. 

the  entire  intestinal  tract  and  especially  the  duodenum  is  attacked,  and  in  monkeys  the 
lower  part  of  the  intestine  is  involved. 

The  associated  nervous  manifestations  are,  according  to  experiments  of  Dopter, 
referred  to  changes  in  the  spinal  cord  itself.  These  are  of  a  nature  similar  to  acute  ante- 
rior poliomyelitis.  Occasionally  a  polio-encephalitis  is  added. 

An  antitoxic  dysentery  serum  is  obtained  by  immunization  of 
Dysentery     horses  and   goats.     Various    methods    have  been  employed 
Serum.       in  its  obtention.     Of  the  older  authors,   Shiga  and  Kruse 
immunized  animals  with  dysentery  bacteria  and  thus  produced 
a  serum  which  possessed  besides  its  bacteriolytic  and  agglutinating  proper- 
ties also  a  weak  antitoxic  action.     Rosenthal,  Todd,  Kraus   and   Doerr 
employed  the  toxin  itself  for  immunization  purposes. 

In  standardization  of  the  serum  the  properties  to  be  determined,  are 
three.  [Kraus  and  Doerr  employ  rabbits  in  this  work.] 

1.  Its  power  of  neutralizing  toxin  in  vitro. — Toxin  and  antitoxin  are  mixed 
in  various  proportions;  the  mixtures  allowed  to  stand  fifteen  minutes  at  room 
temperature  and  then  injected  intravenously. 

2.  Its  power  of  neutralizing  toxin  in  vivo. — The  toxin  is  injected  into  the 
right  vein  and  the  antitoxin  at  the  same  time  into  the  left  vein. 

3.  Its  curative  power. — The  antitoxin  is  injected   at   various  intervals 
after  the  toxin. 

These  three  therapeutic  factors  do  not  appear  simultaneously.  The  power  of  neutral- 
ization in  vitro  is  first  in  evidence.  Only  very  much  later  does  the  serum  develop  its 
curative  strength  and  ability  to  neutralize  in  vivo. 

In  animal  experimentation,  the  antitoxic  serum  exhibits  its  neutralizing  and  curative 
properties  only  in  cases  where  intravenous  injections  are  applied. 

Dysentery  serum  has  been  employed  with  fairly  good  results.  Infections 
caused  by  the  Shiga-Kruse  bacilli  can,  however,  alone  be  benefited.  The 
serum  should  be  given  subcutaneously  and  as  early  in  the  stage  of  the  disease 
as  possible.  The  dose  advised  by  the  different  authors  varies  greatly,  on 
account  of  the  inconstancy  in  strength  of  the  numerous  sera  and  the  severity 
of  the  infection.  In  cases  of  moderate  illness,  it  is  as  a  rule  sufficient  to  give 
one  to  two  injections  of  20  c.c.  of  a  strong  antitoxic  serum  which  can  neutral- 
ize toxin  both  in  vivo  and  in  vitro.  Vaillard  and  Dopter  have  injected  as 
many  as  80  to  100  c.c.  in  the  severer  cases. 

The  good  effect  of  the  serum  manifests  itself  by  an  improvement  in  both 
the  general  and  local  symptoms.  If  high  fever  exists,  the  temperature  sinks. 
If  collapse  temperature  is  present,  it  usually  rises.  The  subjective  com- 
plaints, especially  the  sleeplessness,  improve.  The  blood  in  the  stools  dis- 
appears; the  movements  of  the  bowels  become  less  frequent  and  the  severe 
pains  concomitant  with  the  same  are  absent.  Finally,  the  consistency  of 
the  stools  changes  and  at  the  end  becomes  normal. 

Prophylactic  use  of  the  serum  has  met  very  favorable  confirmation  in  the 


STAPHYLOLYSIN. 


Staphyloly- 
sin. 


work  of  Kruse,  Vaillard  and  Dopter,  and  Rosculet.  Rosculet's  statistics  are 
especially  interesting.  In  1905  during  a  dysentery  epidemic  in  Roumania, 
Rosculet  injected  eighteen  apparently  healthy  individuals  living  at  the  homes 
where  dysentery  cases  existed,  with  5  c.c.  of  the  serum.  Eighteen  similar 
patients  were  removed  from  the  dysentery  surroundings,  but  received  no 
serum.  The  results  were  that  of  the  first  group  no  fresh  cases  of  infection 
arose,  while  of  the  control  group  fourteen  were  infected. 

It  is  rather  premature  to  determine  definitely  the  value  of  the  dysentery 
serum  therapy;  enough  has  been  seen,  however,  to  advocate  its  use  whenever 
possible. 

Staphylolysin,  or  Staphylohemotoxin. — According  to  the  experi- 
ments of  .M.  Neisser  and  Wechsberg  the  pyogenes  staphylo- 
cocci  produce  a  typical  hemolysin  which  is  identical  for  both 
the  aureus  and  albus  cultures.  By  immunization  with  this 
hemotoxin,  an  antihemotoxin  (antitysin)  is  obtained.  Neisser  and  Wechs- 
berg further  discovered  that  serum  both  human  and  of  certain  animal  species 
normally  contained  antistaphylolysin,  less,  however,  in  amount  than  immune 
sera.  Working  on  the  principle  that  in  staphylococcus  diseases,  a  hemo- 
toxin is  formed  which  incites  the.  development  of  antihemotoxin  for  the  pro- 
tection of  the  animal,  Bruck,  Michaelis  and  Schulze  attempted  to  employ 
the  presence  of  antistaphylolysin  in  the  serum  as  evidence  of  the  existence  of . 
Staphylococcus  infections. 

As  staphylolysin,  a  twelve  to  thirteen  day  old  bouillon  culture  of 
freshly  isolated  staphylococcus  pyogenes  serves  very  well.  This  can  be 
preserved  by  adding  5  c.c.  of  the  following  mixture  to  100  c.c.  of  the 
bouillon  filtrate:  10  carbolic,  20  glycerin,  70  aqua.  The  hemotoxin  content 
is  approximated  according  to  the  following  scheme: 


Result  of  hemolysis  after  2  hours 

Amount  of  filtrate. 

Fresh  rabbit  blood. 

Phys.  NaCl. 

in  incubator  at  37°  and  24  hours 

in  ice  box. 

0.2      c.c. 

i  drop. 

ad  2  c.c. 

complete. 

O.I         C.C. 

i  drop. 

ad  2  c.c. 

complete. 

0.05    c.c. 

i  drop. 

ad  2  c.c. 

complete. 

0.025  c.c. 

i  drop. 

ad  2  c.c. 

complete. 

O.OI      C.C. 

i  drop. 

ad  2  c.c. 

incomplete. 

0.005  c-c- 

i  drop.                  ad  2  c.c. 

layer  of  red  blood  cells. 

Thus  0.025  is  the  smallest  dose  which  can  completely  hemolyse  the  given 
quantity  of  red  blood  cells. 

The  amount  of  antilysin  is  estimated  by  adding  varying  amounts  of 
serum  to  the  constant  minimal  hemolytic  dose  of  the  staphylolysin  and 
determining  what  amounts  of  serum  contain  enough  antilysin  to  prevent 


86 


TOXIN  AND  ANTITOXIN. 


hemolytic  action  of  the  staphylolysin.  It  is  best  to  allow  the  staphylolysin 
and  serum  to  remain  mixed  for  some  time  before  adding  the  rabbit  blood,  so 
as  to  give  the  antitoxin  a  chance  to  neutralize  the  toxin. 
i  As  every  normal  serum  contains  a  certain  amount  of  antilysin,  it  is 
necessary  in  order  to  obtain  the  pathological  variations,  to  use  a  normal 
serum  as  a  control.  Such  a  serum,  o.i  of  which  just  suffices  to  neutralize 
twice  the  minimal  hemolytic  dose,  was  dried  in  vacuum  and  used  by  Bruck, 
Michaelis  and  Schulze,  as  standard  serum. 

Estimation  of  antilysin  content  of  the  standard  serum : 


Twice     the     minimum 
hemolytic  toxic  dose. 

0.05 

0.05 

0.05 

0.05 

0.05 

Standard  normal  serum. 

O.2 

O.I 

0.05 

0.025 

O.OI 

Result  after  24  hours..  .  . 

No 
hemolysis. 

No 
hemolysis. 

Slight 
hemolysis. 

Moderate 
hemolysis. 

Complete 
hemolysis. 

These  mixtures  were  allowed  to  stand  for -one  hour  at  37°  C.  and  then  i 
drop  of  rabbit's  blood  was  added,  allowed  to  remain  for  two  hours  at  37° 
and  twenty-four  hours  in  the  ice  box. 

The  standard  serum  was  always  freshly  prepared  in  the  form  of  a  10 
per  cent,  solution  in  distilled  water  (0.1:1). 

In  the  above  manner  the  antilysin  content  of  the  serum  from  patients 
with  distinct  or  suspicious  staphylococcus  infections  was  estimated.  The 
completely  neutralizing  dose  of  the  standard  serum  (o.i  above)  was  taken 
as  i  and  the  neutralizing  dose  of  the  serum  for  examination  compared 
with  this;  if  0.05  c.c.  of  a  serum  x  neutralized  the  same  amount  of  toxin  as 
o.i  of  standard  serum,  the  antilysin  value  of  the  serum  x  was  2. 

From  the  comparative  studies  of  Bruck,  Michaelis  and  Schulze  it  was  con- 
cluded that  most  of  the  normal  sera  had  values  ranging  from  i  down; 
occasionally  results  as  high  as  5  were  obtained.  Out  of  twenty-five  cases  of 
staphylococcus  infections  nineteen  gave  values  varying  from  10  to  100.  Fig- 
ures as  high  as  these  can,  according  these  authorities,  become  of  valuable  interest 
and  aid  in  diagnosis. 

Although  these  findings  were  corroborated  by  Arndt  and  others,  this 
method  cannot  as  yet  be  classed  among  those  of  clinical  diagnostic  impor- 
tance. Similar  study  of  other  infections  has  not  been  undertaken. 

In  addition  to  the  toxins  reviewed  in  these  chapters,  recent  work  has 
proven  that  toxins  may,  under  certain  conditions  be  derived  from  bacteria 
other  than  those  mentioned,  e.g.,  cholera,  typhoid  bacteria  and  meningo- 
cocci.  Problems  such  as  these  are  still,  however,  open  to  scientific  discussion; 
consequently  no  exact  statements  can  be  made  here. 


CHAPTER  DC 

THE  TOXINS  OF  THE  HIGHER  PLANTS  AND  ANIMALS  AND  THEIR  ANTIBODIES. 
FERMENTS  AND  ANTIFERMENTS. 

The  toxins  thus  far  studied  were  all  secretory  products  of  bacteria.  This 
power  of  forming  toxins  is  not,  however,  limited  to  bacteria  alone,  as  there  is  a 
class  of  higher  plants  and  animals  that  produce  characteristic  poisons 
against  which  immunization  can  be  undertaken  and  an  antitoxic  serum 
obtained.  Pollen  toxin  and  snake  poison  are  the  only  members  of  the 
groups  which  bear  any  practical  medical  interest.  The  detailed  study  of 
these  plant  toxins  (Phytotoxin)  and  those  of  animal  origin  (Zootoxin)  has, 
however,  greatly  increased  the  theoretical  knowledge  of  the  phenomena  of 
reaction  and  immunity. 

Phytotoxins. 

The  most  important  phy  to  toxins  are: 

1.  Ricin. 

2.  Abrin. 

3.  Crotin. 

4.  Pollen. 

Ricin  is  a  deadly  poison,  of  which  the  smallest  fractions  of  a  milligram, 

Rjcin.         are  sufficient  to  kill  rabbits.     Like  bacterial  toxins,  ricin  requires  for  its 

action  an  incubation  period  of  at  least  twenty-four  hours.     The  typical 

post-mortem  findings  consist  of  redness  and  swelling  of  Peyer's  patches.     Ricin  is  a 

hemo toxin;  if  mixed,  as  an  emulsion,  with  red  blood-cells,  the  erythrocytes  sink  to  the 

bottom  and  are  agglutinated. 

Ehrlich  succeeded  in  immunizing  animals  against  ricin  by  first  giving  it  to  them  per 
os  in  increasing  doses  for  a  long  period  of  time,  and  later  on  by  subcutaneous  injection. 
The  antitoxic  serum  thus  produced  neutralizes  the  poisonous  action  of  ricin  both  in  vivo 
and  in  vitro. 

Abrin,  a  vegetable  poison,  is  obtained  from  jaquirity  (Abrus  precatorius) 
Abrin.         and  in  its  action  closely  resembles  ricin,  but  is  less  poisonous.     It  is  a 
marked  irritant  of  the  conjunctiva  and  was  at  one  time  employed  in  cases 
of  trachoma. 

Roemer  found  that  by  repeated  instillation  of  abrin  in  to -the  same  conjunctival  sac, 
no  reaction  was  ultimately  obtained  (local  immunity),  while  the  conjunctiva  of  the  other 
eye  retained  its  susceptibility.  If  the  instillation  was  continued  for  a  long  period  of  time,  a 
" general  immunity"  was  attained  which  extended  to  the  conjunctivse  of  both  eyes.  As 
a  result,  in  the  serum  of  such  animals  anti-abrin  could  be  demonstrated. 

87 


88  THE    TOXINS    OF    THE  HIGHER   PLANTS  AND  ANIMALS. 

It  is  the  seed  of  croton  tiglium  that  gives  rise  to  crotin,  a  substance  less 
Crotin.  poisonous  than  either  ricin  or  abrin.  It  does  not  agglutinate,  but  hemo- 

lyses  rabbits'  red  blood  cells.  Toward  the  red  blood-cells  of  other 
species  of  animals  (e.g.,  bird),  it  is  entirely  inactive.  The  immunization  of  rabbits  is 
readily  brought  about  by  subcutaneous  injections.  Their  serum  neutralizes  the  hemo- 
toxic  action  in  vitro. 

The  pollen  toxin  has  been  described  by  Dunbar  as  the  etiologi- 
Hay-fever.  cal  factor  of  hay-fever.  In  Germany  the  disease  seems  to 

come  chiefly  from  pollen  of  the  grasses  and  grains.  (Rye 
pollen  being  most  active) ;  whereas  in  America,  apparently,  the  most  impor- 
tant pollen  springs  from  members  of  the  ambrosia  (rag  weed)  and  solidago 
(golden  rod). 

The  toxin  is  isolated  by  mixing  for  ten  hours  the  ground  pollen  with  5  per  cent.  NaCl 
solution  and  0.5  per  cent,  phenol  at  37°  C.  Then,  in  the  form  of  a  proteid  it  is  precipi- 
tated by  the  addition  of  eight  to  ten  volumes  of  96  per  cent,  alcohol  and  the  resultant 
white  precipitate  dissolved  in  physiological  salt  solution. 

Susceptibility  to  the  pollen  toxin  is  limited  only  to  certain  individuals. 
Some  are  influenced  by  the  rye  pollen  only,  others  by  the  golden  rod  alone, 
while  a  third  class  is  affected  by  all.  The  cause  for  this  peculiar  idiosyn- 
cracy  is  unknown. 

All  those  who  suffer  from  hay-fever  develop  a  marked  conjunctivitis  whenever  even 
the  slightest  amount  of  pollen  proteid  (i/iooo  mg.)  is  dropped  into  the  conjunctival  sac. 
In  addition,  all  the  symptoms  of  hay-fever  or  asthma  may  be  incited.  Similar  effects 
are  in  evidence  when  subcutaneous  injections  are  resorted  to. 

For  purposes  of  immunization  horses  are  most  suitable,  but  only  those 
which  after  an  injection  of  pollen  extracts  manifest  a  local  and  general  reac- 
tion. This  is  found  in  one-third  of  the  cases.  Their  serum  rendered 
immune  is  capable  of  neutralizing  all  effects  of  the  pollen  toxin. 

As  regards  the  standardization  of  this  serum,  it  is  effected  by  mixing  the 
dosis  minima  certe  efficax  of  the  toxin  with  various  dilutions  of  the  serum 
and  instilling  the  mixture  into  the  conjunctival  sac  of  individuals  with  a 
tendency  for  hay-fever.  That  amount  of  the  serum  which  suffices  to  neu- 
tralize the  toxic  ravages  is  taken  as  the  unit  of  measure.  Sera  of  at  least 
thirty  times  the  unit  strength  are  employed. 

The  immune  serum  is  manufactured  in  fluid  and  powder  form  by  Schim- 
mel  &  Co.  of  Miltitz,  near  Leipzig,  and  is  placed  on  the  market  under  the 
name  of  "Pollantin."  Its  use  is  mainly  local,  and  that  by  spraying  a 
small  quantity  of  the  pollen  powder  upon  the  nasal  mucosa  several  times 
daily  and  by  placing  "several  granules  into  the  conjunctival  sac  with  a 
camel's-hair  brush.  The  serum  can  also  be  employed  as  a  prophylactic. 

If  the  eyes  are  especially  reddened,  it  is  best  to  deposit  some  fluid  serum 
into  the  conjunctival  sacs  every  day.  Prausnitz  advises  the  injection  of  i 


SNAKE   POISONS. 


to  2  c.c.  of  the  serum  subcutaneously  when  asthmatic  attacks  occur,  or 
when  the  above  local  treatment  has  failed. 

In  America  a  special  Pollantin  is  made  against  the  frequent  form  of  hay- 
fever  known  as  "  autumn  catarrh"  by  immunization  with  the  pollen  of  the 
golden  rod  and  rag  weed. 

The  pollantin  therapy  and  prophylaxis  has  been  quite  satisfactory,  inas- 
much as  two-thirds  of  the  patients  remain  either  entirely  free  from  attacks 
or  are  so  greatly  benefited  that  their  general  duties  are  not  interfered  with. 
The  only  radical  means  of  curing  the  disease  is  a  change  of  climate,  suitable 
to  the  patient. 


The  Zootoxins. 

Most  important  of  the  animal  toxins  are 
i.  Phrynolysin  (toad  poison),  | 

*      Ararhnnlvsm    ^n?Hprn™n^  Simple  hemotoxins. 


Arachnolysin  (spider  poison), 

3.  Snake  poison, 

4.  Scorpion  poison,  Lecithm 


5.  Bee  poison, 


The  one  striking  characteristic  of  toxins,  that  an  immunity  can  be 
raised  against  them,  is  also  possessed  by  these  poisons.  Beyond  this  fact 
they  present  many  variations  from  the  true  class  of  toxins.  Most  of  these 
poisons  are  complex,  i.e.,  they  contain  more  than  one  toxin,  and  all  are 
hemotoxic. 

Toad  poison  is  produced  by  rubbing  up  the  skins  of  the  Bombinator  igneus;  spider 
poison  by  trituration  of  the  living  "cross  spiders"  (Epeira  diadema)  in  three  or  four  times 
the  amount  of  physiological  salt  solution  containing  toluol. 

The  toad  and  spider  poisons  contain  simple  hemotoxins,  that  is  to  say,  by  the  mixture 
of  small  amounts  of  this  toxin  with  erythrocytes  absolutely  serum-free,  hemolysis  of  the 
latter  takes  place.  Not  all  species  of  blood  are  affected  alike.  The  red  blood  corpuscles 
of  sheep,  goats,  and  rabbits  are  especially  adapted  for  experiments  with  phrynolysin, 
while  rabbits',  rats',  and  human  blood  is  more  suitable  for  arachnolysin.  Immunity  of 
rabbits  is  easily  attained. 


Snake-poisons. 

The  most  familiar  poisonous  snakes  are  the  Cobras  (Naja)  of  India  and 
Indo-China  which  belong  to  the  family  of  Colubridae,  the  European  viper, 
and  the  American  rattlesnake;  the  last  two  being  of  the  Viperidae  species. 
The  poisons  of  these  two  families  show  great  individual  differences.  Thus, 
those  of  the  Colubridae  group  are  decidedly  thermo-resistant  (temperatures 


90  THE    TOXINS    OF   THE  HIGHER   PLANTS   AND   ANIMALS. 

as  high  as  100°  C.)  while  the  viper's  poison  is  entirely  destroyed  at  a  tem- 
perature varying  between  80  to  85°  C.,  and  markedly  weakened  at  70°. 

Snake  poisons,  as  a  rule,  produce  both  local  reactions  at  the  point  of  the 
bite,  and  severe  general  disturbances. 

The  cobra  bite  is  only  slightly  painful.  A  characteristic  feeling  of  stiffness  extends 
from  the  point  of  infection  over  the  entire  body.  In  several  hours  a  rapidly  increasing 
weakness  sets  in  terminating  in  deep  coma  and  death. 

The  viper  bite  incites  a  very  strong  local  reaction.  The  point  of  infection  is  red, 
extremely  painful  and  swollen.  Convulsions  and  hemorrhages,  followed  by  delirium 
which  finally  changes  into  stupor  are  manifest,  and  death  takes  place  in  one  to  three  days. 
If  the  poison  gets  into  the  circulation  directly,  death  is  likely  to  occur  in  a  few  minutes. 

The  prognosis  of  a  snake  infection  depends  largely  upon  the  situation  of 
the  bite.  The  greater  the  blood  supply  of  the  infected  area  the  more  dan- 
gerous is  the  result.  Bites  received  through  the  clothing  are  relatively  less 
dangerous,  as  a  great  part  of  the  poison  remains  adherent  to  the  clothing. 

Snake  poisons  act  primarily  upon  the  nervous  system  and  blood,  although 
they  exhibit  a  number  of  other  toxic  and  ferment  properties.  Thus  viper 
toxin  occasions  immediate  coagulation  of  the  blood  by  its  action  upon  the 
vascular  endothelium  and  has  for  this  reason  been  called  by  Flexner  and 
Noguchi,  "Hemorrhagin." 

Furthermore  all  snake  poisons  have  a  hemolytic  power. 

Cobra  hemolysis  represents  one  of  the  most  interesting  of 

Cobra       biological  phenomena,  and  as  it  may  possibly  be  employed 
Hemolysis.    in  clinical  methods  of  examination  its  action  will  be  here 
reviewed. 

Cobra  hemotoxin  is  characterized  by  its  power  of  dissolving  the  red  blood 
corpuscles  of  certain  kinds  of  animals  (ox,  sheep  and  goat)  only  in  the 
presence  of  serum.  Other  red  blood  cells  do  not  require  any  serum  for 
their  hemolysis  (dog,  guinea-pig,  man,  rabbit,  horse).  If  the  red  blood 
corpuscles  of  the  first  group  of  animals  washed  free  of  their  serum  are  mixed 
with  cobra  poison,  no  hemolysis  takes  place.  On  subsequent  addition  of 
any  fresh  serum,  hemolysis  is  in  evidence.  (Flexner,  Noguchi). 

The  agent  which  instigates  the  hemolytic  substance  belongs  undoubt- 
edly to  the  class  of  lipoids.  Of  these,  lecithin  stands  pre-eminent.  It  is, 
however,  by  no  means  certain  whether  that  is  the  only  or  the  most  important 
activator. 

Some  sera  exhibit  this  activating  influence  only  when  first  heated.  In 
their  unheated  state  they  are  entirely  inactive.  Other  sera  act  in  a  manner 
decidedly  the  reverse.  Kyes  and  Sachs  mention  that  this  depends  alto- 
gether upon  the  nature  of  the  lecithin  union  in  respect  to  the  other  elements 
present.  The  following  table  shows  the  various  combinations  and  their  re- 
sultant action. 


COBRA  HEMOLYSIN   TEST. 


Combination. 

Power  of  serum  activation. 

Serum. 

Red  cells. 

Unheated. 

Heated  at 

56° 

65  to  100° 

Horse.                       OT.  .  . 

+ 
+ 
+ 

+ 
+ 
+ 
+ 

+ 

+ 

+ 
+ 

+ 
+ 

+ 
+ 
+ 
+ 
+ 

+  weak  hemolysis. 

+ 
+ 
+ 
+ 
+ 

Horse 

Horse 

Ox  

Horse  

Ox  ..   .. 

Ox 

Sheep  
Sheep  . 

Ox  

Sheep  .   . 

Human  

Ox  

Human  

Human.  . 

Rabbit  
Guinea-pig  

Ox 

Ox  

Guinea-pig 

Rabbit 

+  Signifies  hemolysis. 
—  Signifies  no  hemolysis. 


Cobra  Hemolysin  Test. 

1.  Washing  of  Erythrocytes. — The  blood  is  collected  into  sterile  flasks  containing 
sterile  glass  beads.     It  is  then  shaken  and  thus  defibrinated  to  prevent  coagulation. 
The  defibrinated  blood  is  next  centrifugalized  and  the  serum  separated  and  drawn  off 
by  means  of  a  pipette.     The  red  blood  cell  sediment  is  then  mixed  with  physiological 
salt  solution  and  again  centrifugalized.     This  procedure  is  repeated  several  times  until 
all  the  serum  is  removed.     The  red  blood-cells  as  used  are  in  a  5  percent,  suspension; 
i.e.,  i  part  of  washed  erythrocytes  suspended  in  19  parts  of  saline. 

2.  The  Activating  Agent. — In  order  to  obtain  an  activating  agent  0.2  of  serum  or  a 
o.i  per  cent,  of  a  lecithin  solution  is  employed.     The  lecithin  can  be  kept  as  a  stock 
solution  consisting  of  i  g.  lecithin  in  100  c.c.  of  methyl  alcohol.     Ao.i  per  cent,  solution 
of  the  stock  mixture  is  made  by  mixing  o.i  c.c.  of  the  solution  with  9.9  c.c.  of  physiological 
salt  solution. 

3.  The  snake  poison,  hemotoxin,  is  resistant  toward  heat  so  that  it  may  be  heated  to 
almost  70°  C.  without  interfering  with  its  activity.     Cobra  poison  contains  the  greatest 
amount  of  hemotoxin.     While  i  mg.  of  cobra  toxin  hemolyses  i  c.c.  of  5  per  cent,  horse's 
red  blood-cells  in  five  to  ten  minutes,  a  similar  amount  of  viper  toxin  requires  thirty 
minutes  for  the  same  action. 

V.  Dungera  and  Coca  explain  this  type  of  hemolysis  on  the  ground  of  the 
existence  of  a  ferment  within  the  snake  poison  which  breaks  up  the  lecithin 
with  the  liberation  of  oleic  acid.  This  acid  has  long  been  known  as  a 
hemolytic  agent.  The  necessity  for  adding  lecithin  or  serum  to  certain 
species  of  blood  is  explained  by  the  variability  in  the  lecithin  content  of  the 
erythrocytes. 


92  THE   TOXINS    OF   THE  HIGHER   PLANTS  AND  ANIMALS. 

Calmette  notes  in  the  blood  of  tuberculous  patients  more  than  the  normal 

Cobra  Toxin  amount  of  lecithin;  for  that  reason  their  serum  can  be  used  in  very  small 

Activation  in  doses  to  activate  the  cobra  hemolysin.     By  this  means  he  has  found  it 

Tuberculosis,  possible  to  attain  a  diagnostic  reaction  for  tuberculosis.     On  examination 

of  the  blood  of  177  tubercular  individuals  he  has  found: 

78  per  cent,  of  positive  reactions  in  the  first  stage  of  tuberculosis. 
57  per  cent,  of  positive  reactions  in  the  second  stage  of  tuberculosis. 
70  per  cent,  of  positive  reactions  in  the  third  stage  of  tuberculosis. 

Szaboky  has  confirmed  these  findings,  but  not  enough  control  examinations  of  normal  in- 
dividuals or  of  other  infections  have  been  made  to  firmly  establish  the  diagnostic  value 
of  the  test. 

The  hemolysis  of  snake  poison  can  be  overcome  or  interfered  with  by  the 
addition  of  large  amounts  of  normal  serum,  cholesterin,  and  small  amounts 
of  snake  poison  serum. 

Much  and  Holzmann  have  recently  described  the  so-called  "Psycho- 

The  Psycho-   reaction"  which  can  be  explained  thus — Normal  serum,  when  added  to  a 

reaction.       mixture  of  cobra  extract  and  human  red  blood-cells  will  not  interfere 

with  consequent  hemolysis.  If,  however,  the  serum  obtained  is  from 
patients  suffering  from  depressive  mania,  circular  insanity  or  dementia  pracox,  and 
added  to  the  cobra  extract  and  human  red  blood-corpuscles,  the  expected  hemolysis  does 
not  take  place.  One  would  naturally  suppose  that  this  fact  would  be  employed  for 
clinical  diagnosis,  but  unfortunately  it  has  been  generally  proven  by  most  authorities  in 
this  line  that  it  is  altogether  impossible  to  do  so  for  the  simple  reason  that  it  is  not  abso- 
lutely specific.  Bauer  has  found  the  same  reaction  with  navel  blood.  It  is  probable 
that  the  interference  with  hemolysis  is  brought  about  by  an  increase  in  the  cholesterin 
of  the  serum — a  possibility  in  diseases  of  the  central  nervous  system  more  so  than  under 
any  other  physiological  or  pathological  conditions. 

In  immunizing  laboratory  animals  one  cannot  start,  at  the 

beginning  at  least,  with  inoculations  of  the  unaltered  snake 
Immunity. 

poison. 

Phisalix  and  Bertrand  begin  by  employing  subcutaneous  injections  of  a  toxin  heated  to 
75°  C.  and  after  two  days  use  one-half  of  the  minimal  lethal  dose  of  the  unaltered  toxin. 

Calmette  weakens  the  cobra  poison  by  the  addition  of  an  equal  amount  of  i  per  cent, 
gold  chlorid,  and  after  four  such  injections  with  increasing  amounts  at  each  time,  the  pure 
toxin  in  very  small  doses  is  employed. 

In  the  same  manner  Calmette  immunized  horses  and  obtained  highly 
antitoxic  sera.  He  tested  the  strength  of  these  sera,  as  follows: 

1.  Upon  Rabbits. — Each  animal  received  an  injection  of  2  c.c.  of  the  serum  into  the 
vein  of  one  ear,  and  after  two  hours  i  mg.  of  toxin  into  the  vein  of  the  other  ear.     A 
control  animal  was  similarly  treated  with  toxin  only.     The  latter  animal  died  in  a  half 
hour,  while  the  former  remained  alive. 

2.  Upon  White  Mice. — Diminishing  amounts  of  serum  were  mixed  in  test  tubes, 
with  o.oooi  gm.  of  toxin  (in  i  per  cent,  solution)  and  the  mixtures  injected  into  the 
mice.     The  greatest  amount  of  serum  which  completely  neutralizes  the  toxin  must  be 
0.03  c.c. 


PAROXYSMAL  HEMOGLOBINURIA. 


93 


According  to  Calmette  one  can  approximate  the  efficiency  of  an  immune 
serum  by  its  antihemolytic  power  inasmuch  as  the  hemotoxic  and  neurotoxic 
actions  run  parallel.  This  is  denied  by  Noguchi. 

The  scorpion  and  bee  poisons  display  properties  similar  to  those  of  the 
cobra  poison.  They  also  combine  with  lecithin  to  produce  hemolysis. 

Thus  far,  it  has  been  shown  that  the  lipoids,  especially  lecithin  are 
actively  associated  in  the  hemolysis  of  erythrocytes;  whether  the  toxin  com- 
bines with  the  lipoids  and  forms  a  toxolipoid  (toxolecithid)  which  is  hemo- 
toxic, or  whether  as  v.  Dengen  believes,  the  hemolytic  action  is  due  to  the 
fatty  acid  derived  from  the  lecithin  by  the  ferment  action  of  substances 
contained  in  the  poison,  has  not  been  definitely  proven. 

Quite  recently  it  has  been  thought  that  pernicious  anemia  and  parox- 
ysmal hemoglobinuria  are  closely  associated  with  such  toxolipoids. 

Tallquist  obtained  from  a  Bothriocephalus  latus,  a  hemotoxic 
Pernicious     poison  of  a  lipoid  nature  which  experimentally  produced  a 

Anemia.      blood  picture  characteristic   of  pernicious  anemia.     But  it 
would  be  incorrect  to  associate  all  forms  of  pernicious  anemia 
with  tape  worm  poison;  more  probable  is  it  that  hemotoxins  are  formed 
within  the  organism  itself. 

In  paroxysmal  hemoglobinuria  a  hemotoxin  of  very  peculiar 
Paroxysmal    properties  is  found  circulating  in  the  blood. 
Hemoglobi-    It  can  be  demonstrated  as  follows. 

miria.  Im  EhrlicWs  method.— One  of  the  patient's  fingers  is  ligatured 
by  means  of  a  small  tourniquet  and  kept  immersed  in  ice-cold 
water  for  a  half  hour.  Some  blood  is  then  collected  into  a  capillary  pipette 
from  the  finger  thus  tied,  and  as  a  control,  blood  from  a  finger  of  the  other 
hand  is  drawn  off.  This  is  allowed  to  clot  and  then  centrifugalized.  The 
results  are  that  the  serum  from  the  finger  held  in  the  ice  water  is  tinged  red 
from  dissolved  hemoglobin  while  the  control  serum  is  normally  pale. 

2.  Donath-Landsteiner's  method  repeats  Ehrlich's  experiment  in  vitro. 
The  patient's  and  the  control  individual's  serum  are  each  mixed  with 
washed  human  erythrocytes  in  various  proportions.  It  does  not  matter 
whether  the  red  blood  cells  are  obtained  from  the  patient  or  normal  individ- 
ual. The  mixtures  are  allowed  to  remain  for  one-half  to  one  hour  in  the 
ice  box  and  then  from  one  to  three  hours  at  a  temperature  of  37°  C.  The 
serum  from  the  paroxysmal  hemoglobinuria  patient  shows  hemolysis. 

A  control  tube  containing  the  same  ingredients,  in  the  same  proportions 
and  maintained  at  either  cold  or  warm  temperatures,  but  not  at  both  in 
succession  as  above,  exhibits  no  hemolysis. 

The  hemolytic  process  in  this  disease  is  of  a  complex  nature.  In  the 
cold,  one  element  combines  with  the  erythrocytes,  and  at  high  temperature 
another  unfolds  hemolytic  tendencies.  Some  sera  lacking  or  not  having 
enough  of  the  second  element  in  the  serum,  demonstrate  no  hemolysis. 


94  THE    TOXINS    OF   THE   HIGHER   PLANTS  AND  ANIMALS. 

But  on  addition  of  some  normal  serum  hemolysis  occurs.  It  can  therefore 
be  concluded  that  the  second  factor  which  acts  in  the  heat  is  present 
within  normal  serum,  while  the  first  substance,  the  specific  one,  is  found 
only  in  the  blood  of  those  suffering  from  paroxysmal  hemoglobinuria ;  (and 
according  to  Donath  and  Landsteiner  in  10  per  cent,  of  cases  of  general 
paralysis).  It  is,  in  addition,  the  author's  opinion,  that  similar  toxic 
substances  exist  in  the  blood  of  epileptics  and  idiots. 

Not  all  cases  of  paroxysmal  hemoglobinuria  possess  this  characteristic 
hemotoxin.  In  some  it  is  only  found  periodically. 

No  explanation  has  as  yet  been  offered  for  these  varying  phenomena. 
Attempts  have  been  made  to  ascertain  whether  the  hemotoxin  is  stimulated 
by  an  external  agent  or  by  infection  (Lues,  malaria,  trypanosomiasis)  or 
whether  it  is  of  endogenous  origin.  The  answer  is  still  for  the  future  to 
disclose. 

The  Antiferments. 

Ferments  are  very  closely  allied  to  toxins  in  their  biological  structure. 
By  the  immunization  of  animals  with  ferments  in  as  pure  a  form  as  possible, 
antiferments  can  be  demonstrated.  Just  like  antitoxins,  antiferments  can 
neutralize  their  respective  ferments  in  vitro.  As  to  their  presence,  it  is  quite 
important  to  know  that  they  are  found  in  normal  serum  in  certain  small 
quantities  (together  with  antitoxins).  The  difference  in  their  presence  in 
a  normal  serum  and  that  in  an  immune,  is  purely  a  quantitative  one. 
The  antiferments  thus  far  demonstrated  are 

Antilabferment.  Antipepsin. 

Antitrypsin.  Antisteapsin. 

Antifibrinferment. 

It  is  difficult  to  obtain  by  immunization  an  antiferment  serum  of  very  high 
strength.  Probably  the  normal  organism  is  so  regulated  that  it  compensates 
any  increased  amount  of  antiferment. 

Till  recent  times  the  demonstration  of  antiferments  bore  no  clinical 
Antitrypsin.    interest.     The  antibodies  of  the  proteolytic  enzymes  first  began  to  attract 
attention  when  the  inhibitory  influence,  which  blood  serum  has  upon  the 
autolysis  of  organs  was  proven.     It  was  Jochmann  and  Miiller  who  showed  in  connection 
with  their  studies  of  the  proteolytic  ferments  of  leucocytes,  that  apart  from  these,  the 
serum  itself  possesses  an  inhibitory  influence  upon  the  leucocyte  ferment.     This  is  found 
to  be  especially  marked  in  diseases  associated  with  great  destruction  of  leucocytes.      Fol- 
lowing them,  Marcus,  as  well  as  Brieger  and  Trebing  discovered  a  restraining  influence  in 
the  serum  upon  the  action  of  pancreas  trypsin  and  proved  that  the  so-called  antitrypsin 
was  considerably  increased  in  carcinoma  patients.     Bergmann  and  Meyer,  working  also 
along  these  lines,  then  demonstrated  that  the  wrongly  called  "  carcinoma 
Brieger's       reaction"  was  by  no  means  specific  for  carcinoma,  but  was  found  in  a 
Cachexia      large  number  of  other  diseases.     It  cannot,  as  Brieger  later  announced, 
Reaction.      even  be  considered  as  a  criterion  for  cachexia  (cachexia  reaction). 

Undoubtedly,  the  already  normal  antiproteolytic  power  of  the  serum 


THE  ANTIFERMENTS.  95 

can  be  considerably  increased  in  animal  experimentation  by  a  group  of  well-known  pro- 
teolytic  agents,  and  especially  by  leucocyte  ferment  and  pancreatic  trypsin.  To  differ- 
entiate between  antileucocyte  and  antitrypsin  ferment  in  the  narrow  sense  of  the  word,  is 
impossible.  The  one  "immune  serum"  (sit  venia  verbo,  if  one  can  speak  of  immune 
serum  in  this  sense)  neutralizes  the  other  antigen.  Clinically  a  high  antitryptic  titer  of 
the  serum  is  found  in  about  90  per  cent,  of  carcinoma  patients,  and  is  almost  regularly 
observed  in  infections  with  high  fevers  as  typhoid,  severe  articular  rheumatism,  sepsis, 
etc.  In  pneumonia  there  is  found  during  the  infection  a  marked  change  from  an  exces- 
sively high  to  a  low  titer.  In  Morbus  Basedow  (as  well  as  in  experimental  thyroid  feed- 
ing) it  is  almost  the  rule  to  find  a  high  antitrypsin  content,  but  one  must  always  keep  in 
mind  that  even  few  normal  individuals  show  a  similar  increase. 

The  clinical  diagnostic  import  of  the  antitrypsin  titer  is  slight  in  comparison  with  its 
experimental  increase.  In  accord  with  the  findings  in  Basedow,  and  in  thyroid  feeding 
it  may  be  considered  as  an  outcome  of  increased  proteid  destruction  (hyper-produc- 
tion of  proteolytic  ferments  in  the  tissues?)  Leucocyte  ferment  has  been  found  of  prac- 
tical use  in  the  treatment  of  cold  abscesses,  i.e.,  in  processes  where  leucocytosis  and  failure 
to  produce  polyneuclear  leucocyte  ferment  is  present.  On  the  other  hand  antitrypsin 
or  antileucocyte  ferment  or  even  normal  serum  is  employed  to  counteract  inflammatory 
processes,  i.e.,  to  neutralize  the  excessive  production  of  the  leucocyte  ferments,  with  appa- 
rent success  (Leucoantifermentin,  on  the  market).  According  to  recent  findings,  the 
antitrypsin  titer  of  the  mother's  blood  increases  markedly  during  the  period  of  labor, 
while  that  of  the  fetus  remains  unaltered. 

There  are  two  methods  for  the  antitrypsin  determination.  The  first  was 
devised  by  Jochmann  and  Miiller  for  proving  the  presence  of  leucocyte 
ferment  and  its  antiferment,  and  then  similarly  employed  by  Marcus  in  the 
study  of  pancreatic  ferments.  Its  principle  depends  upon  the  digestive 
action  of  proteolytic  ferments  upon  serum  albumin.  When  a  drop  of 
trypsin  is  placed  upon  a  Loffler's  serum  plate,  after  a  little  while,  a  clear 
spot  appears  where  the  trypsin  was  brought  into  contact  with  the  plate. 
If  to  this  trypsin,  an  amount  of  serum  is  previously  added,  which  fully 
neutralizes  the  digestive  action,  no  clear  zone  appears  upon  Loffler's  plate. 

The  details  of  this  procedure  are  as  follows:  The  ferment  solution  consists  of  o. i  gm. 
trypsin,  well  shaken  with  5  c.c.  of  undiluted  glycerin  and  5  c.c.  of  distilled  water,  then  left 
in  an  incubator  for  a  half  hour  at  55°  C.,  then  again  shaken  and  filtered. 

The  serum  is  mixed  in  small  test  tubes  or  upon  a  glass  slide  with  varying  amounts  of 
the  trypsin;  thus  i  loopful  of  serum  is  mixed  with  1/2,  i,  2,  3,  4,  etc.,  up  to  20  loopfuls  of 
the  trypsin  solution  and  of  each  of  these  mixtures  one  loopful  is  placed  upon  Loffler's 
plate.  (Ox  serum  plate  should  be  three  days  old).  The  plates  are  then  placed  into  the 
incubator  for  twenty-one  hours  at  55°  C.  The  presence  or  absence  of  the  clear  zones 
determines  the  quantities  of  ferment  which  respectively  have  not  or  have  been  neutralized 
by  the  one  drop  of  serum  (e.g.,  i  :  6  means  that  in  the  mixture  of  6  loopfuls  of  trypsin  and  i 
loopful  of  the  serum  for  examination  the  digestive  power  of  the  trypsin  was  still  interfered 
with). 

The  inequality  in  the  strength  of  the  Loffler  plates,  their  variability  in  the  degree  of 
alkalinity,  the  measurement  by  loopfuls,  all,  might  prove  to  be  sources  of  error  which 
may  greatly  influence  the  results.  Thus  the  latter  can  only  be  taken  as  approximate, 
relative  values. 


96  THE   TOXINS    OF   THE  HIGHER   PLANTS  AND  ANIMALS. 

The  second,  more  exact  and  satisfactory  method  was  introduced  pri- 
marily by  Gross  and  Fuld  for  presenting  the  action  of  trypsin,  and  was  modified 
by  v.  Bergmann  together  with  Bamberg  and  Meyer  for  the  determination 
of  antitrypsin.  Numerous  workers  have  found  it  thoroughly  reliable. 
Its  principle  is  based  upon  the  digestion  of  a  clear  casein  solution.  If  the 
entire  amount  of  casein  is  digested,  no  more  is  left  to  be  precipitated  by  the 
addition  of  acid  and  therefore  the  solution  remains  clear.  If,  however, 
casein  has  been  left  undigested,  the  addition  of  acid  will  produce  a  turbid 
solution  or  even  a  white  precipitate. 

The  necessary  reagents  are: 

1.  Casein  Solution. — One  gm.  of  casein  is  dissolved  under  slight  heating  in  100  c.c.  of 
N/io  Na  O  H;  this  solution  is  next  neutralized  by  N/io  HC1,  litmus  being  used  as  indi- 
cator, and  diluted  with  physiological  salt  solution  up  to  500  c.c.     (If  sterilized,  it  can  be 
kept  for  a  long  while). 

2.  Trypsin  Solution. — 0.5   gm.  of   trypsin   (purissimum   Griibler)   is   dissolved   in 
50  c.c   of  NaCL-f  0.05  c.c.  of  normal  sodium  hydrate  solution  and  then  diluted  with 
physiological  saline  up  to  500  c.c. 

3.  Acid  Solution. — Five  c.c.  of  acetic  acid+  45  c.c.  of  alcohol +  50  c.c.  of  water. 

First  the  titration  of  the  trypsin  solution  is  undertaken  in  order  to  find  out  how  much 
trypsin  is  required  to  fully  digest  a  constant  quantity  of  casein.  Gradually  increasing 
amounts  of  trypsin  (from  o.i  to  0.6  c.c.)  are  placed  into  six  test-tubes  and  to  each  2.0  c.c. 
of  casein  are  added.  These  tubes  are  placed  into  an  ncubator  at  37°  for  one-half  hour, 
and  then  several  drops  of  the  acid  solution  are  placed  into  each  tube.  The  first  tube,  and 
all  those  above  it  that  remain  absolutely  clear,  contain  enough  trypsin  to  fully  digest  the 
2.0  c.c.  of  casein. 

Now  comes  the  second  part  of  the  test. 

Into  each  of  eight  to  ten  test-tubes,  are  placed  2  c.c.  of  the  casein  solution  and  0.5 
c.c.  of  a  2  per  cent,  dilution  of  the  serum  for  examination;  to  these  is  next  added  the  tryp- 
sin solution  in  successively  increasing  amounts,  beginning  with  the  smallest  quantity 
which  in  the  first  part  of  the  test  was  sufficient  to  completely  digest  the  given  amount  of 
casein.  Salt  solution  is  then  added  to  each  of  the  test  tubes  so  that  all  contain  an  equal 
quantity  of  fluid,  and  the  mixtures  placed  into  an  incubator  at  37°  for  one-half  hour.  At 
the  end  of  this  time,  several  drops  of  the  acid  are  added  to  each  tube.  Those  tubes  which 
become  cloudy  or  show  a  precipitate,  designate  the  amounts  of  trypsin  solution  which  have 
been  neutralized  by  the  0.5  c.c.  of  diluted  serum.  For  example: 

In  the  first  part  of  the  test  it  was  found  that  the  tube  containing  0.4  c.c.  of  trypsin 
was  the  first  to  remain  clear,  in  other  words  was  sufficient  to  fully  digest  2  c.c.  of  the  casein 
solution.  In  the  second  part  of  the  test  the  lower  limit  of  the  added  trypsin  dilution  was 
0.4,  and  it  was  found  that  the  tubes  containing  0.4,  0.5,  0.6  and  0.7  c.c.  of  trypsin,  for 
example,  now  gave  precipitates  and  only  0.8  remained  clear.  This  indicates  that  part  of 
the  formerly  sufficient  amount  of  trypsin  was  now  neutralized  by  the  antitrypsin  of  the 
added  serum  so  that  digestion  was  interfered  with.  Thus  the  antitrypsin  titer  in  this  case 
is  0.8. 

Recently  the  above  method  of  trypsin  titration  has  been  applied  to  the  determination 
of  the  presence  of  pancreatic  ferment  in  the  intestine,  feces,  and  stomach  contents. 


CHAPTER  X. 
AGGLUTINATION. 

If  the  serum  from  an  immunized  animal  or  a  patient  convalesc- 
The  Pheno-    mg  after  an  infection,  be  mixed  with  a  suspension  of  the  bac- 

menon  of  teria  which  were  involved  in  the  production  of  said  conditions, 
Agglutination.  a  peculiar  phenomenon  takes  place.  In  the  formerly  dif- 
fusely cloudy  liquid,  small  granules  and  clumps  appear  which 
sink  to  the  bottom  of  the  test-tube  and  leave  a  supernant  clear  liquid.  On 
microscopic  examination,  the  sediment  presents  bacteria  (which  have  re- 
mained alive  as  is  demonstrable  by  making  cultures  of  same).  This  same 
observation  can  be  made  when  the  experiment  is  performed  in  a  hanging 
drop  with  perhaps  more  flattering  results.  The  bacteria  are  seen  to  lose 
their  motility,  adhere  to  each  other,  finally  gravitate  toward  larger  groups 
and  arrange  themselves  in  clumps.  The  phenomenon  thus  described  was 
discovered  by  Gruber  and  Durham,  and  is  called  agglutination;  while  sub- 
stances which  cause  this,  agglutinins. 

If  instead  of  the  immune  or  that  of  the  convalescent  patient,  normal 
serum  is  employed,  and  the  above  test  repeated,  it  will  be  seen  that  agglutina- 
tion likewise  occurs.  The  reaction  here  is,  however,  somewhat  incom- 
plete, the  clumps  smaller,  and  formed  much  more  slowly.  //  a  quantitative 
determination  with  different  dilutions  of  both  sera  is  made,  the  power  of 
agglutination  disappears  with  the  normal  serum  at  a  low  dilution,  while  the 
immune  serum  remains  perfectly  active  at  even  much  greater  dilutions. 
Thus  the  main  difference  between  the  agglutinating  normal  and  immune 
serum  is  a  quantitative  one  depending  upon  the  amount  of  agglutinins 
present.  Whether  any  qualitative  difference  exists  between  the  normal  and 
immune  agglutinins  is  doubtful.  It  is,  however,  of  no  practical  significance. 

If  instead  of  homologous  bacteria,  different  (heterologous)  bacteria  are 
employed,  e.g.,  cholera  vibrio  and  typhoid  serum,  agglutination  also  takes 
place,  if  the  typhoid  serum  is  used  in  concentrated  or  only  slightly 
diluted  form;  but  in  moderate  or  great  dilutions,  no  agglutination  occurs. 
Normal  serum  will  agglutinate  the  cholera  vibrio  in  the  same  strength  as  the 
immune  typhoid  serum.  In  other  words  the  typhoid  serum  contains  more 
agglutinins  for  its  homologous  bacteria,  than  a  normal  serum,  but  it  has  only 
the  same  titer  of  agglutination  as  a  normal  serum  for  heterologous  bacteria. 

The  agglutination  reaction  is  specific  in  the  respect  that  weak  dilutions  of 
7  97 


98  AGGLUTINATION. 

serum  will  agglutinate  only  its  homologous  bacteria  and  leave  the  heterologous 
ones  uninfluenced.  Agglutination  becomes  non-specific,  when  concentrated  or 
strong  dilutions  of  serum  are  employed. 

The   relative    specificity    just   described  is  of  great  clinical 
Diagnostic    diagnostic  value.     For  example,  given  a  serum  suspicious  of 

Value  of      typhoid   the  question  is  to   establish  this   absolutely.     One 

Agglutination,  immediately   proceeds   to   make   a   suitable   dilution   of   the 

unknown  serum  and  mixes  with  it  known  typhoid  bacilli. 

A  similar  dilution  of  normal  serum  is  also  made  as  a  control  and  mixed  with 

the  same  amount  of  typhoid  bacilli.     If  agglutination  occurs  with   the 

unknown  serum  and  not  with  the  control  serum,  the  former  must  have  come 

from  a  typhoid  patient.     If  the  bacteria  are  not  agglutinated,  the  serum  was 

not  of  typhoid  origin. 

In  an  equal  manner  can  the  identity  of  unknown  bacteria  be  established 
by  the  use  of  known  sera.  Thus,  when  certain  bacteria  have  been  isolated 
and  information  is  wanted  as  to  whether  same  are  of  a  typhoid  nature,  an 
emulsion  of  these  is  made  and  mixed  with  a  typhoid  serum  in  suitable 
dilution,  and  a  similar  amount  of  bacteria  is  mixed  with  a  normal  serum  of 
like  dilution.  Agglutination  occurring  in  the  first  of  these  mixtures  and 
absent  in  the  second,  proves  the  typhoid  character  of  the  unknown  bacteria. 
In  this  manner  the  agglutination  test  can  be  used  for  identification  of  an 
antigen. 

The  practical  application  of  agglutination  has  been  greatly  made  use  of 
in  cases  of  typhoid  fever.  Here  the  production  of  agglutinins  is  very  easily 
stimulated  in  the  course  of  the  disease  and  generally  they  can  be  demon- 
strated in  the  serum  seven  to  ten  days  after  infection.  The  agglutinins  re- 
main not  only  during  the  active  stage  of  the  disease,  but  also  during  the 
convalescing  period.  Widal,  the  Parisian  clinician,  was  the  first  to  adopt 
this  agglutination  reaction  for  the  serum  diagnosis  of  typhoid.  It  is  thus 
commonly  known  as  the  Widal  reaction. 

The  technique  of  the  reaction  is  as  simple  as  its  principle. 

jc  mque  >    r^g  accounts  for  its  wide  adoption.     It  may  either  be  per- 


Agglutmation.  .  .  .  . 

formed  macroscopically  or  microscopically  (orientation  test). 


The  Macroscopic  Agglutination  Reaction. 

The  necessary  requirements  for  carrying   out   the   reaction  consist  of: 

1.  The  immune  serum  and  a  normal  control  serum; 

2.  A  homogeneous  bacterial  emulsion. 

The   production   of   a   homogeneous   bacterial    emulsion   offers    slight 
technical  difficulties. 

It  can  be  obtained  in  the  following  ways  : 

a.  Bouillon  Culture.  —  Many  bacteria,  like  typhoid,  paratyphoid,  dysen- 


MACROSCOPIC  AGGLUTINATION   REACTION. 


99 


tery,  coli,  etc.,  grow  very  easily  in  broth.  Such  a  fresh  (twenty-four  hours), 
diffusely  turbid  culture  can  be  employed  readily  for  agglutination  purposes. 
In  place  of  live  bacteria,  dead  may  also  be  used — a  fact  which  has  greatly 
added  to  the  practical  application  of  the  test. 

For  obtaining  the  latter,  0.5  per  cent,  of  phenol  or  i  per  cent,  of  formalin  (40  per 
cent.)  are  added  to  the  twenty-four  hour  bouillon  cultures.  The  result  is,  that  a 
sediment  of  bacteria  is  formed  from  which  the  supernatant  fluid  should  be  carefully 
poured  off.  The  bacterial  suspension  is  kept  on  ice  and  thoroughly  shaken  before  use. 

Ficker  has  in  this  way  prepared  standard  emulsions  of  dead  typhoid  and  paratyphoid 
bacilli  which  are  sold  by  Merck  under  the  name  of  "Ficker's  Diagnosticum." 

For  carrying  out  Widal's  test,  a  small  quantity  of  the  patient's  blood  is  collected 
into  a  capillary  tube  and  the  end  closed  with  sealing-wax.  The  blood  is  allowed  to  clot, 
and  the  serum  to  separate  off.  The  separation  of  the  latter  can  be  hastened  by  centri- 
fugalization. 

In  practice,  the  Widal  test  as  performed  with  Ficker's  diagnosticum,  is  arranged  as 
follows: 


Bacillus  suspension. 

Dilution  of  serum. 

Physiological  salt  solution. 

Tube  i   o  c;  c  c 

o  c  c  c 

Tuhp  n    o   c  r  r 

o  ^  c  c  of  i  "  10 

Tiihe  7    o   c  r  r 

o  ^  c  c  of  i  '  ^o 

j.  uuc  ^,  u  .  ^  c.c. 
Tnhp  A    o    c  r  r 

o  ^  c  c  of  i  *  100 

One  of  four  results  may  be  obtained. 


Positive  reaction. 

Doubtful  reaction. 

Negative  reaction. 

Worthless 
reaction. 

i.  No  agglutination  
2.  Marked  agglutination.  .  .  . 

3.  Marked  agglutination.  .  .  . 

No  agglutination  
Marked  agglutination.  . 

Very  slight  agglutina- 
tion. 
No  agglutination  

No  agglutination  
Very  slight  agglutina- 
tion. 
No  agglutination  

No  agglutination  

Agglutination. 
Agglutination. 

Agglutination. 
Agglutination. 

It  is  very  advisable  to  make  control  tests  also  with  normal  serum.  After  the  mixture 
of  the  various  ingredients  the  tubes  are  placed  into  the  incubator  at  37°  for  two  hours. 
Then  the  results  are  read  off  and  the  first  tube  must  show  absolutely  no  agglutination 
otherwise  (as  seen  in  Division  No.  4  above),  the  entire  test  is  of  no  significance.  The 
cause  for  such  spontaneous  or  pseudo-agglutination  occurring  in  tube  i,  may  be 
found  either  in  the  bacterial  emulsion  or  NaCl  solution.  The  grade  of  agglutination 
is  estimated  by  the  size  of  the  agglutinated  clumps  and^the  rapidity  with  which  they 
are  formed.  The  mild  grades  of  agglutination  are 


IOO  AGGLUTINATION. 

For  typhoid  a  positive  reaction  is  one  where  agglutination  takes  place  in  the  dilution  of 
i  :  100;  a  positive  result  in  the  dilution  of  i  :  50  can  only  be  considered  as  probably 
positive. 

As  has  been  said,  broth  cultures  may  be  used  for  the  agglutination  test  if  the  bacteria 
grow  diffusely  and  regularly  within  the  bouillon.  This  is  not  the  case,  however,  with  all 
bacteria,  as  for  example,  the  cholera  vibrio  which  produces  a  thin  pellicle  upon  the  sur- 
face of  the  broth. 

b.  Agar  Cultures. — Kolle  and  Pfeiffer  have  advised,  instead  of  broth  the 
use  of  agar  cultures.  The  bacteria  are  washed  off,  and  an  even  emulsion 
made  in  physiological  salt  solution,  or  in  a  dilution  of  the  serum  for  exam- 
ination. 

The  details  of  the  procedure  are  as  follows: 

Into  a  row  of  test-tubes  is  placed  i  c.c.  of  various  dilutions  of  the  serum  for  examina- 
tion, e.g.,  i  :  10,  i  :  50,  i  :  100,  i  :  200,  i  :  500.  A  normal  serum  is  similarly  diluted  as 
a  control.  In  this  respect  only  the  higher  concentrations  of  the  normal  serum  are  neces- 
sary. One  other  test-tube  is  to  contain  i  c.c.  of  saline  only. 

A  full  loop  of  an  eighteen  to  twenty-four  hours'  old  agar  culture  is  evenly  and  finely 
rubbed  up  in  each  of  the  above  test-tubes  as  follows: 

The  test-tube  is  held  almost  horizontally  in  the  left  hand  between  the  thumb  and 
index  finger;  a  platinum  loop  between  the  thumb  and  index  finger  of  the  right  hand  is 
filled  with  the  bacteria  from  the  agar  culture,  and  placed  into  the  tube  containing  the 
serum  dilutions.  The  bacteria  are  then  gently  and  thoroughly  rubbed  up  upon  the 
moistened  wall  of  the  tube  but  not  within  the  fluid.  By  rolling  the  test-tube  slightly,  a 
part  of  the  rubbed  up  bacteria  is  washed  into  the  fluid  and  the  remaining  bacterial  mass 
is  again  triturated.  This  process  is  repeated  until  all  the  bacteria  are  washed  into  the  fluid. 
Thus,  a  homogeneous  suspension  is  obtained. 

The  author  has  found  this  method  of  Pfeiffer  and  Kolle  most  accurate. 

It  is  worthy  of  note  in  this  connection,  that  the  controls  show  no  clumps 
or  granules.  (Pseudo-agglutination).  There  are  some  bacteria  which 
can  be  evenly  emulsified  only  with  great  difficulty,  while  others  are  very 
easily  agglutinated  even  by  normal  serum.  In  either  case  the  test  is  not 
conclusive. 

For  the  hanging  drop  method,  blood  is  collected  into  a  Wright  capsule  or  a 
small  test-tube;  6  to  8  drops  of  blood  suffice.  The  blood  is  allowed  to  clot 
or  the  serum  is  hastened  by  centrifugalization.  Four  loopfuls  of  broth  or 
saline  (or  equal  amounts  as  measured  by  a  Wright  pipette)  are  placed  upon 
each  of  two  slides.  To  one  of  these  one  loopful  of  the  serum  (or  one  equal 
part  as  measured  by  the  Wright  pipette)  is  added  and  thoroughly  mixed. 
From  this  mixture  one  loopful  or  equal  measure  is  mixed  with  the  broth  or 
normal  saline  upon  the  second  slide;  thus  making  serum  dilutions  of  i :  5 
upon  the  first  slide  and  1:25  upon  the  second  slide.  A  loopful  of  typhoid 
culture  is  placed  upon  the  center  of  each  of  two  cover  slips.  To  the  first  is 
added  one  loopful  of  the  serum  dilution  i:  25,  and  to  the  second  is  added 
one  loopful  of  the  serum  dilution  i :  5  thus  making  a  dilution  of  i :  50  and 
y  :'IQ  respectively.  ;'Eachr  cover  slip  is  inverted  over  a  hollow  slide  protected 


GROUP  AGGLUTINATION.  IOI 

by  vaseline,  and  examined  microscopically.     A  control  with  normal  serum 
and  also  one  with  culture  alone  should  be  made. 

For  the  identification  of  bacteria  only  highly  agglutinating 
Production  of  animal  sera  can  be  employed.     Rabbits,  goats  and  horses, 
Agglutinating  are  most  suitable  for  such  experiments.     The  best  results 
Sera.         are  obtained  when  the  animals  are  immunized  intravenously 
by  repeated  injections  with  gradually  increasing  doses  of  dead 
bacteria  (killed  at  60°  C.).     Usually  two  to  three  injections  of  1/4  to  i  agar 
culture  suffice   to  give  an  agglutinating  titer  of  i  to   5000.     The  serum 
should  be  withdrawn  eight  to  ten  days  after  the  last  injection.     As  a  matter 
of  course  the  titer  of  the  serum  should  be  tested  from  time  to  time,  because 
the  height  of  the  titer  curve  can  only  reach  a  certain  point.     When  a  suffi- 
cient strength  is  obtained  the  animal  is  bled.     It  is  not  possible  to  produce 
equally  strong  agglutinating  sera  for  all  bacteria. 

The  agglutinins  belong  to  the  class  of  the  more  resistant  serum  sub- 
stances. With  slight  addition  of  carbolic  acid  they  can  be  preserved  on  ice 
for  a  long  time.  Heating,  variously  affects  the  different  bacterial  agglu- 
tinins. The  agglutinins  for  pest  and  tubercle  bacilli  are  destroyed  at  56°  C. 
while  other  bacteria  are  not  influenced  by  even  higher  temperatures.  The 
animal  from  which  the  agglutinating  serum  has  been  obtained  also  influences 
to  a  great  degree,  the  resistance  towards  heat.  Thus  the  typhoid  agglu- 
tinating serum  derived  from  the  horse  is  much  more  resistant  than  that 
obtained  from  the  rabbit. 

The  Microscopic  (Orientation)  Agglutination  Test. 

This  method  is  especially  of  use,  when  only  small  amounts  of  culture 
or  serum  are  obtainable.  Also,  if  agglutination  is  employed  for  the  quick 
recognition  of  bacteria,  as  for  example,  when  it  is  desirable  to  know  whether 
a  blue  colony  on  a  Conradi-Drigalski-agar  plate  is  typhoid  or  not. 

In  such  a  case  a  drop  of  the  immune  serum  in  the  dilution  of  1 150  or  i  :ioo 
is  placed  upon  a  cover-glass  held  with  a  Cornet's  forceps,  and  a  small  part  of 
the  bacterial  colony  for  identification  carefully  mixed  with  this  serum.  As 
controls,  a  mixture  is  made  with  salt  solution  and  with  normal  serum.  If 
agglutination  occurs,  small  granules  or  clumps  can  readily  be  seen  with  the 
naked  eye  by  holding  up  the  cover-glass  against  the  light.  The  control 
glasses  on  the  other  hand  should  show  only  a  homogeneous  turbidity. 
These  changes  are  still  more  evident  if  the  mixture  is  examined  micro- 
scopically in  the  form  of  a  hanging  drop.  (Described,  p.  100.) 

Group  Agglutination. 

On  testing  the  titer  of  a  strongly  agglutinating  typhoid  serum,  and  a 
strongly  agglutinating  cholera  serum,  against  typhoid,  paratyphoid,  colon  and 
cholera  bacteria,  the  results  will  appear  to  be  the  following: 


102  AGGLUTINATION. 


Agglutination  titer. 

Of  typhoid  serum. 

Of  cholera  serum. 

Against  typhoid 

I    '    2OOO 

I    '    IO 

Against  paratyphoid  

I    '.    IOO 

i  :  10 

Against  bacter.  coli  

i  :  2Z 

i  :  10 

Against  cholera      

i  :  10 

i  :  -?ooo 

The  cholera  serum  acts  strictly  in  accordance  with  the  rules  stated  above 
for  specific  agglutinins,  i.e.,  marked  agglutination  with  homologous  bac- 
teria; very  weak,  with  heterologous.  The  typhoid  serum  on  the  other  hand, 
although  in  the  main  it  fulfills  the  same  requirements,  nevertheless  mani- 
fests some  important  differences  when  mixed  with  heterologous  bacteria. 
It  has  practically  no  influence  upon  the  cholera  vibrio  with  which  the 
typhoid  bacillus  is  not  at  all  related;  its  agglutination  of  i  :  10  can  be  attained 
even  by  a  normal  serum.  The  colon  bacillus  which  closely  resembles  the 
typhoid,  morphologically,  but  which  has  very  different  biochemical  proper- 
ties is  more  strongly  agglutinated,  i  :  25;  while  the  paratyphoid  bacillus, 
very  much  like  the  typhoid  bacillus,  both  morphologically  and  biologically 
is  agglutinated  even  in  larger  dilutions,  i  :  100.  This  entire  phenomenon,  is 
an  expression  of  the  biological  relationship  of  the  various  bacterial  groups 
and  is  known  as  group  reactions. 

An  understanding  of  group  reactions  is  to  be  found  in  a  more 
.  ,  .  .  complete  conception  of  specificity.  From  this  source  we  have 

learned  that  the  difference  in  antibodies  is  influenced  by  the 
dissimilarity  of  the  injected  antigen.  For  example,  the  difference  between 
the  cholera  and  typhoid  agglutination  is  caused  by  that  existing  in  the 
protoplasmic  structure  of  the  respective  bacteria.  As  these  bacteria,  how- 
ever, are  not  constituted  of  a  distinct  chemically  defined  substance,  but 
made  up  of  a  mixture  of  various  substances  there  may  be  a  number 
amongst  them  which  can  act  as  antigens.  If,  figuratively  speaking,  there 
are  five  different  elements  in  the  body  of  the  typhoid  bacillus  which  can 
act  as  agglutinogens,  i.e.,  antigens,  these  should  be  able  to  form  according  to 
the  law  of  specificity  five  different  agglutinins.  On  mixing  a  typhoid  serum 
with  typhoid  bacilli,  one  brings  together  five  distinct  antigen  antibody  com- 
binations and  consequently  complete  and  thorough  action  results  of  this 
union.  A  biological  relationship  of  bacteria  implies  the  existence  of  some 
common  protoplasmic  constituents.  Expressed  in  the  same  figurative 
manner  the  colon  bacillus  can  be  said  to  have  antigen  number  i  in  common 
with  the  typhoid  and  paratyphoid  bacillus  and  the  paratyphoid  may  have 
antigen  numbers  i  and  2  in  common  with  the  typhoid  bacillus.  As  a  result, 
the  typhoid  serum  will  react  with  colon  bacilli  by  virtue  of  their  common 


CASTELLANl'S    TEST.  103 

agglutinin  number  i,  and  with  paratyphoid  bacilli  through  its  agglutinins 
numbers  i  and  2.  The  other  "partial  agglutinins"  remain  inactive  on 
account  of  the  missing  suitable  agglutinogens. 

The  existence  of  such  partial  antigens  and  partial  antibodies  is,  for  some 
bacteria  more  than  of  mere  theoretical  importance.  It  is  even  possible  that 
a  strong  colon  serum  will  agglutinate  no  colon  bacilli  other  than  that  partic- 
ular strain  employed  for  the  production  of  the  serum.  Such  being  the 
case  with  a  number  of  micro-organisms,  the  sera  made  at  present  both  for 
diagnostic  and  therapeutic  purposes  are  polyvalent  (multipartial).  By 
polyvalent  serum  is  meant  one  which  is  produced  either  by  immunizing 
animals  with  many  different  strains  of  the  same  bacterium,  or  a  mixture  of 
sera  obtained  from  different  animals  immunized  with  various  strains. 

The  practical  importance  of  partial  agglutinins  is  recognized 
Castellani's    in  the  diagnosis  of  mixed  infections.     Castellani  found  that  by 
Test.         the  mixture  of  an  immune  serum  with  its  corresponding  bac- 
teria, the  agglutinins  for  these  as  well  as  the  partial  agglu- 
tinins for  the  heterologous  bacteria  are  absorbed.     On  the  other  hand,  if  the 
same  serum  be  mixed  with  the  heterologous  bacteria,  the  agglutinins  for  the 
homologous  group  are  quantitatively  retained. 

A  practical  example  will  make  this  clearer. 

The  serum  of  a  patient  agglutinates  typhoid  as  well  as  paratyphoid  bacilli,  in  a  dilu- 
tion of  i  :  100.  This  may  indicate  one  of  three  possibilities: 

a.  Patient  is  infected  with  typhoid,  but  has  formed  an  exceptionally  large  number  of 
partial-agglutinins  for  paratyphoid  bacilli. 

b.  Patient  is  infected  with  paratyphoid  bacilli,  but  has  formed  at  the  same  time  many 
partial-agglutinins  for  typhoid. 

c.  Patient  has  a  mixed  infection  of  typhoid  and  paratyphoid  and  therefore  formed 
agglutinins  for  both. 

A  decision  in  regard  to  the  above  may  be  reached  according  to  the  following  method 
given  by  Castellani: 

Four  rows  of  test  tubes  are  arranged,  each  row  containing  three  tubes  with  i  c.c.  of 
serum  dilutions  i  :  10,  i  :  50,  i  :  100  respectively.  In  each  of  the  first  and  second  row, 
i  loopful  of  typhoid  bacteria  is  emulsified. 

In  each  of  the  third  and  fourth  row,  i  loopful  of  paratyphoid  B.  bacilli  is  emulsified. 
The  tubes  are  placed  into  the  incubator  for  two  hours,  absence  or  presence  of  agglu- 
tination in  each  test-tube  noted,  and  after  centrifugalization  (which  may  become  unneces- 
sary if  the  bacteria  are  strongly  clumped  or  grouped  at  the  bottom  of  the  tube),  the  super- 
natant liquid  is  transferred  into  other  test-tubes  and  kept  in  the  same  order. 
Then  each  of  the  first  row  receives  i  loopful  of  typhoid  bacilli, 

each  of  the  second  row  receives  i  loopful  of  paratyphoid  B.  bacilli, 
each  of  the  third  row  receives  i  loopful  of  typhoid  bacilli, 
each  of  the  fourth  row  receives  i  loopful  of  paratyphoid  B.  bacilli, 
All  are  once  more  placed  into  the  incubator  for  two  hours. 

a.  If  typhoid  exists,  the  agglutination  titer  in  the  second  part  of  the  test  will  become 
weaker  in  the  first,   second  and  fourth  rows,   while  that  in  the  third  row  remains 
the  same. 

b.  If  paratyphoid  exists,  the  titer  for  typhoid  in  the  first  and  third  row  becomes  less, 


104  AGGLUTINATION. 

that  for  paratyphoid  in  the  fourth  row  diminishes,  while  the  titer  in  the  second  row  for 
paratyphoid  remains  the  same. 

c.  If  a  mixed  infection  exists,  the  agglutination  titer  in  the  first  and  fourth  row  dimin- 
ishes and  in  the  second  and  third  row  remains  the  same. 

In  this  connection  a  few  exceptions  may  be  mentioned: 

A  serum  which  is  kept  for  a  long  time,  frequently  loses  part 
Agglutinoids.  or  even  all  of  its  agglutinating  titer.     Whereas  it  formerly 
agglutinated  in  the  strength  of  i :  1000,  it  may  now  become 
inactive  in  dilutions  even  of  i :  10.     The  first  thought  that  arises  in  explana- 
tion of  this  is  that  the  serum  has  perhaps  degenerated  and  the  aggtutinins 
destroyed.     If,  however,  further  dilutions  are  made,  i :  100  may  show  mild, 
while  i :  500  strong   agglutination.     This,  first  of   all,  demonstrates   that 
agglutinins  are  still  present,  although  diminished  in  amount,  and  second, 
that  another  substance  has  arisen  which  in  the  stronger  concentrations 
interferes  with  agglutination.     A  simple  experiment  explains  this. 

If  the  test-tube  containing  the  serum  dilution  i  :  10  and  the  non-agglutinated  bacteria 
be  centrifugalized,  the  serum  removed  and  the  bacteria  mixed  with  a  known  strongly 
agglutinating  serum,  it  will  be  found,  that  the  bacteria  have  become  inagglutinable. 
Substances  of  certain  kinds  have  combined  with  the  bacteria  and  prevented  them  from 
undergoing  agglutination.  These  substances  are  strongly  specific,  acting  only  upon 
homologous  bacteria.  Their  origin  can  also  be  demonstrated. 

An  agglutinating  serum  which  is  heated  to  65°  or  70°  C.  loses  its  agglutinating  power 
but  the  substance  interfering  with  the  subsequent  agglutination  has  remained.  Ehrlich 
explains  the  situation  as  follows :  He  claims  that  agglutinins  are  built  complexly ;  that  they 
possess  a  binding  (haptophore)  group  by  means  of  which  they  unite  with  the  bacteria 
(agglutinogen)  and  a  second  group  (ergophore  or  agglutinophore)  by  virtue  of  which 
agglutination  results.  If  serum  is  kept  for  a  long  period  of  time,  or  exposed  to  high 
temperature,  many  of  the  ergophore  groups  are  rendered  inactive,  while  the  haptophore 
groups  being  more  resistant  remain  with  their  full  potentialities,  and  unite  with  bacteria. 
Agglutinins  possessing  only  their  haptophore  groups  are  known  as  agglutinoids.  They 
combine  with  the  bacteria,  and  still  do  not  agglutinate  them,  but  at  the  same  time  prevent 
other  agglutinins  from  acting.  If  this  old  agglutinoid  and  agglutinin  containing 
serum  is  diluted,  so  few  of  both  of  these  substances  remain  that  the  bacteria  can 
absorb  both,  allowing  the  relatively  few  agglutinins  to  manifest  their  activity. 

It  is  important  to  note  in  this  respect,  that  occasionally  even  a  fresh,  highly  valent 
serum,  will  present  a  tendency  towards  interfering  with  the  agglutination  processes. 
This  is  also  explained  by  the  existence  of  agglutinoids — a  fact  as  yet  not  definitely  proven. 

Another  finding,  only  encountered  in  exceptional  cases,  is  the  existence 
of  the  so-called  non-agglutinable  strains  of  bacteria.  These  give  all  the 
characteristics  of  the  general  class  of  bacteria  to  which  they  belong,  but  are 
not  agglutinated  by  their  respective  serum;  as,  for  example,  a  strain  of 
typhoid  bacilli,  which  are  not  agglutinated  by  any  typhoid  serum.  The 
only  positive  proof  that  they  are  typhoid  bacilli,  is  the  ability  to  produce  by 
their  employment  an  active  immunity  against  fully  virulent  typhoid  bacteria. 

Non-agglutinable  strains  of  bacteria  can  be  isolated  especially  from  the  lower  animals. 
At  times,  however,  they  regain  their  agglutination  property  when  they  are  grown  in 


AGGLUTINATION   IN    TYPHOID  AND   PARATYPHOID.  105 

artificial  media  and  frequently  subplanted.  Possibly,  the  reason  that  the  bacteria  become 
inagglutinable  at  all,  is  that  they  undergo  immunization  within  the  organism  against  the 
existing  agglutinins.  By  growing  bacteria  in  agglutinating  serum  for  a  certain  time,  one 
can  obtain  inagglutinable  strains. 

i.  Agglutinins  for  typhoid  and  paratyphoid  A  and  B,  can,  not 

Agglutination  infrequently,  be  demonstrated  in  the  patient's  serum  as  early 

in  Typhoid    as  the  third  day,  but  as  a  rule,  at  about  the  beginning  of  the 

and  Para-     second  week  of  the  disease.     Moreover,  they  remain  within 

typhoid.  tne  serum  for  several  weeks  after  the  illness  and  disappear 
only  gradually.  A  positive  agglutination  test  does  not,  how- 
ever, mean  the  existence  of  the  corresponding  disease.  A  healthy  bacillus 
carrier  can  also  have  an  agglutinating  serum.  Some  cases  of  icterus  catarrhalis 
even  give  a  positive  Widal  test.  But  in  order  to  assign  to  this  last  a  correct 
explanation,  one  must  remember  that  typhoid  bacilli  may  remain  in  the  gall- 
bladder for  years  and  thus  lead  to  catarrhal  inflammation  and  stone  formation. 

Partial  agglutinins  from  coli  infections  must  always  be  considered. 
Some  authorities  mention  a  positive  Widal,  in  connection  with  endocarditis 
maligna,  sepsis,  malaria,  phthisis,  and  miliary  tuberculosis. 

An  absence  of  the  agglutination  test,  especially  at  an  early  part  of  the 
illness,  should  not  influence  a  negative  diagnosis  of  typhoid  too  greatly, 
inasmuch  as  many  cases  are  known  where  the  reaction  appeared  for  the 
first  time  during  the  period  of  convalescence.  In  the  employment  of  this  test 
as  an  aid  for  the  differential  diagnosis  between  several  bacterial  infections, 
it  is  best  to  titrate  the  serum  to  its  limit,  as  the  higher  titer  for  one  class  of 
bacteria  generally  speaks  in  favor  of  the  infection  by  the  same.  Para- 
typhoid serum  agglutinates  typhoid  bacilli  only  slightly,  while  true  typhoid, 
both  typhoid  and  paratyphoid  bacteria  with  equally  high  force.  In  severe 
and  difficult  cases,  Castellani's  test  should  be  performed.  Paratyphoid 
B.  serum,  always  gives  the  limit  of  its  agglutinating  titer  both  with  the 
pathogenic  mouse  typhoid  and  hog  cholera  bacillus. 

2.  Cholera.— Only  rarely  has  the  agglutination  test  been  employed  with 
the  serum  of  patients  thus  afflicted.     On  the  other  hand,  the  identification 
of  cholera  suspicious  colonies  in  the  stool  is  regularly  conducted  by  means  of 
this  test.     For  this  purpose,  it  is  very  specific,  as  group  reactions  almost 
never  take  place.     Strongly  agglutinating  sera  are  easily  obtained  by  immu- 
nization of  animals. 

3.  Epidemic,  Cerebrospinal  Meningitis.— Agglutination  in   this  disease 
serves  mainly  for  the  identification  of  suspicious  meningococcus  cultures. 
As  has  been  shown  by  Wassermann  and  Kutscher,  some  strains  are  agglu- 
tinated  only  after  a  long  period  (twenty-four  hours)  and  at  higher  tem- 
peratures as  56°  C.* 

*  Frequently,  during  even  the  first  days  of  the  disease,  the  patient's  serium  in  a  dilution  of 
i-io  gives  the  agglutination  test.  This  is  rare  with  higher  dilutions  of  the  serum  as  1-50.  It 
usually  takes  some  time  before  the  agglutination  becomes  evident. 


106  AGGLUTINATION. 

4.  Dysentery. — The  agglutination  property  is  employed  both  for  testing 
the  serum,  and  identifying  cultures.     The  Flexner  type  of  bacillus,  produces 
agglutinins  more  readily  than  that  of  Shiga-Kruse.     They  are  also  agglu- 
tinated more  readily.     Only  positive  reactions  in  dilutions  of  i :  30,  are  of 
diagnostic  consideration.     Occasionally  partial  agglutination  takes  place 
with  heterologous  dysentery  strains,  typhoid  and  colon  bacteria. 

5.  Pest. — The  reaction  is  very  specific,  but  of  slight  significance,  as  it  appears  only 
upon  the  ninth  day;  occurring  with  a  serum  dilution  of  i  :  3,  it  is  considered  of  positive 
diagnostic  value. 

6.  Malta  Fever. — In  most  instances  the  serum  gives  the  agglutination  reaction  with  the 
micrococcus  melitensis.     Normal  serum  may  give  the  reaction  in  dilution  i  :  30,  so 
that  higher  dilutions  only  are  of  aid  in  diagnosis. 

7.  Staphylo,  Strepto  and  Pneumococci. — Clinically,   the  agglutination  test  is  never 
employed  in  these  cases. 

8.  Tuberculosis. — Here  the  agglutination  test  is  associated  with  the  difficulty  of  obtain 
ing  a  homogeneous  tubercle  bacillus  suspension.     This,  however,  is  overcome  by  one  of 
two  ways. 

a.  Arloing-Courmont's  Method  (1898). — The  tubercle  bacilli  are  obtained  in  the  so- 
called  "homogeneous  culture"  form.     S.  Arloing  first  grows  the  bacteria  upon  potatoes  for 
a  long  time,  and  then  transplants  them  into  glycerin  bouillon  which  is  agitated,  daily  for 
five  minutes.     After  a  number  of  subcultivations,  a  culture  is  finally  obtained  after  several 
months.     This  grows  rapidly  in  a  few  days  and  diffusely  clouds  the  broth. 

Such  a  culture  diluted  with  physiological  saline  solution,  is  used  for  the  test.  Here 
small  test-tubes  are  preferable  and  the  ingredients  should  be  mixed  after  the  following 
proportions : 

2  drops  of  serum  +  10  drops  of  culture  (i  :  5) 
i  drop   of  serum  +  10  drops  of  culture  (i  :  10) 
i  drop   of  serum  +  15  drops  of  culture  (i  :  15) 
etc. 

The  combined  substances  are  well  shaken  and  placed  into  an  incubator.  According  to 
Arloing  and  Courmont,  a  positive  reaction  even  in  the  dilution  of  i  :  5  speaks  for  tuber- 
culosis. Best  results  are  by  this  means  obtained  in  incipient  and  mild  tubercular  cases; 
those  which  are  farther  advanced  do  not  react. 

b.  Method  of  Koch. — Koch  filters  the  ordinary  tubercle  bacillus  bouillon  cultures,  dries 
the  remnants  upon  the  filter,  and  rubs  them  up  in  an  agate  mortar  with  N/5o  NaOH 
up  to  a  dilution  of  i  :  100.     The  solution  is  centrifugalized  and  enough  weak  HC1  is 
added  until  the  reaction  is  only  slightly  alkaline.     The  dilution  is  then  brought  up  to  i  : 
3000  by  the  addition  of  0.5  per  cent,  phenol  in  normal  saline,  and  kept  for  twenty-four 
hours  in  the  incubator. 

A  somewhat  simpler  procedure  is  to  dilute  new  tuberculin  B.  E.  to  i  :  100  with  0.5 
per  cent,  of  carbolic  saline  solution,  centrifugalize  this  for  six  minutes  and  then  dilute  to 
i  :  1000.  The  solution  thus  obtained  can  be  preserved  in  the  ice-box  for  fourteen  days. 
Just  before  using,  a  still  further  dilution  of  i  :  10  is  made. 

The  agglutination  test  has  not  been  generally  adopted  as  a  method  for  diagnosis. 
The  technique  is  rather  difficult,  and  the  results  not  absolutely  reliable.  The  reason  for 
the  latter  is  that  high  agglutination  values  are  rarely  met  with,  and  slight  ones  are  found 
even  in  normal  individuals.  Then,  too,  the  methods  of  tuberculin  diagnosis  are  so  much 
simpler,  that  they  have  been  given  the  preference. 


HEMAGGLUTININ.  107 

Koch  himself  advised  the  agglutination  test,  not  as  a  means  of  diagnosis,  but  rather 
as  an  aid  in  tuberculin  therapy.  He  found  that  during  the  treatment  of  tuberculosis 
with  new  tuberculin  the  agglutinative  power  of  the  patient's  serum  increased.  He 
therefore  took  this  as  an  index  of  the  acquired  immunity.  Further  study,  however, 
convinced  him  that  the  agglutination  cannot  thus  be  interpreted,  so  that  at  the  present 
day  tuberculosis  agglutination  has  no  practical  application. 

10.  Glanders. — Highly  valent  sera  can  be  obtained,  according  to  Kleine,  by  intravenous 
immunization  of  donkeys  and  goats.  The  serum  serves  for  identification  of  the  glanders 
bacilli.  Kleine  prepares  a  standard  bacterial  emulsion  in  the  following  manner:  Four 
well  grown  glanders  cultures  are  killed  at  60°  C.  and  the  mass  of  bacteria  triturated  in 
2  c.c.  of  1/2  per  cent,  carbolic-saline  solution.  This  is  then  diluted  in  a  measuring  glass 
so  that  40  to  50  c.c.  of  carbolic-saline  solution  are  added  for  each  culture.  The  entire 
mixture  is  filtered  through  paper  and  3  c.c.  are  used  in  each  test-tube.  Normally,  horses 
may  have  an  agglutination  titer  up  to  i  :  400.  Glanders  infected  animals  react  as  high  as 
i  :  2000.  Injections  of  mallein  increase  the  agglutination  titer.  Experiences  in  this 
respect  with  the  human  being  are  still  scanty. 

Just  as  injections  of  bacteria  produce  bacterial  agglutinins, 

Hemagglu-    injections  of  erythrocytes  stimulate  the  formation  of  hem- 

tinin.        agglutinins  which  cause  the  red  blood  cells  to  congregate  in 

clumps. 

At  times  the  presence  of  hemagglutinins  is  masked  by  the  simultaneous 
existence  of  hemolysins  which  dissolve  the  red  blood  corpuscles.  If,  however, 
the  immune  serum  is  heated  to  56°  C.  the  hemolysin  is  destroyed,  thus  allow- 
ing the  agglutinins  to  exhibit  their  action.  In  other  instances  as  during  the 
immunization  of  rabbits  with  dog's  erythrocytes,  hemagglutinins  are  formed 
in  such  great  quantities  that  in  mixing  the  immune  rabbit's  serum  with  the 
dog's  erythrocytes  so  strong  an  agglutination  occurs  that  the  hemolysins  can 
no  longer  attack  the  clumped  erythrocytes.  The  hemolysin  presence  can 
be  demonstrated  only  if  clumping  is  prevented  mechanically,  by  thorough 
shaking  of  the  mixture. 


CHAPTER  XL 
PRECIPITINS. 

In  the  former  chapter,  the  phenomenon  of  agglutination  was  explained 
as  a  clumping  of  bacteria  occurring  when  a  serum  is  mixed  with  its  correspond- 
ing bacteria.  In  1897  R.  Kraus  described  a  phenomenon,  very  closely 
allied  to  the  one  just  mentioned.  He  found  that  when  an  immune  serum, 
for  example,  of  cholera,  typhoid,  or  pest,  is  mixed  with  the  clear,  sterile 
nitrate  of  the  respective  bouillon  cultures  of  their  bacteria  (instead  of  the 
bacteria  themselves),  the  clear  solution  becomes  turbid,  and  a  precipitate 
forms.  This  reaction  is  known  as  precipitation,  the  elements  within  the 
immune  serum,  precipitins;  while  the  substances  (antigen)  with  which  the 
precipitin  reacts  and  which  originally  stimulated  the  production  of  the 
precipitin,  precipitinogen. 

Like  all  biological  reactions,  the  phenomenon  of  precipitation  is  not 
limited  to  bacterial  immune  sera  and  culture  nitrates,  but  is  observed  when 
any  animal,  vegetable  or  bacterial  soluble  proteid  substance,  is  mixed  with 
the  serum  of  an  animal  which  has  been  immunized  against  the  particular 
proteid  material  in  question. 

Tschistowitsch  and  Bordet  were  the  first  who  called  attention  to  these  non-bacterial 
precipitins.  Bordet  (1899)  found  that  the  blood  serum  of  rabbits  treated  with  the  serum  of 
chickens  gave  a  specific  precipitate  when  mixed  with  chicken  serum.  Tschistowitsch 
demonstrated  a  similar  reaction  with  the  sera  of  rabbits  treated  with  horse's  and  eel  serum. 

The  biological  structure  of  the  precipitins  is  strongly  analogous  to  that 
of  agglutinins.  Many  authorities,  in  fact,  consider  them  identical.  Whatever 
has  been  said  in  regard  to  the  effects  of  heating  and  addition  of  acids  or 
alkalies  upon  agglutinins,  applies  equally  to  precipitins.  Moreover,  they 
also  are  composed  of  two  groups,  a  binding  (haptophore)  and  a  functionally 
active  (ergophore)  group.  If  the  latter  is  missing,  they  are  known  as 
precipitinoids,  and  can  interfere  with  precipitation  just  as  agglutinoids  do 
with  agglutination. 

In  speaking  of  precipitation,  it  has  always  been  customary  to  differen- 
tiate between  bacterial  and  proteid.  For  practical  purposes  this  division 
is  superfluous  inasmuch  as  the  bacterial  precipitins  are  nothing  more  than 
precipitins  of  bacterial  proteids. 

1 08 


BACTERIAL   PRECIPITIN   REACTIONS.  109 

Bacterial  Precipitin  Reactions. 

For  the  production  of  precipitating  sera,  animals  are  immu- 

Productionof  mzed  either  with  the  bacterial  bodies  themselves,  or  fluids 

Precipitin     containing    the  bacterial    proteid    (precipitinogen) ,   such  as 

Sera.         the  nitrates  of  bouillon  cultures  and  the  various  forms  of 

bacterial  extracts.     The  serum  from  individuals  undergoing 

an  infection  or  convalescing  from  one,  contains  precipitins  against  the 

respective  infective  agent. 

Inasmuch  as  the  reaction  consists  of  the  formation  of  a  precipitate,  it  is 
important  that  both  of  the  ingredients  (precipitin  and  precipitinogen)  be  abso- 
lutely dear  and  have  no  tendency  to  spontaneously  become  turbid,  or  form  a 
precipitate. 

In  order  to  get  a  clear  serum  one  should  avoid  withdrawing 
Obtaining     the  blood  during  the  period  of  digestion  of  the  animal,  because 
Clear  Sera,     it  is  chylous  at  such  a  time.     In  man  the  best  occasion  for 
obtaining  the  blood  is  in  the  morning  before  breakfast.     As 
for  animals,  it  is  advisable  to  give  them  no  solid  food  (or  milk)  for  twenty- 
four  hours  previous  to  venesection.     Then  a  very  minute  quantity  of  blood 
is  withdrawn  and  immediately  centrifugalized  in  order  to  ascertain  whether 
the  serum  is  clear  or  not.     If  it  is  satisfactory,  larger  amounts  may  be  col- 
lected.    Erythrocytes  and  bacteria  produce,  at  some  times,  turbid  serum. 
Simple  sedimentation  or  centrifugalization  suffices  to  overcome  this. 

If  in  spite  of  these  precautions  turbidity  still  persists,  recourse  may  be 
had  in  filtration  through  paper  or  bacterial  niters,  preferably  new  ones. 
This  method  should,  however,  be  used  as  a  last  resort,  because  filtration 
always  tends  to  diminish  the  strength  of  a  serum. 

Bacterial  precipitinogens  are  prepared  by  filtration  either  of 
Bacterial      bouillon  cultures  or  bacterial  extracts.     The  filtrates  must  be 
Precipitin-     absolutely  clear;  also  sterile,  as  frequently  the  reaction  requires 
ogens.        a  long  period  of  time.     If  bacteria  are  present  they  may  grow 
quickly,   and  produce  turbidity.     The    precipitinogen    loses 
after  a  time  its  property  of  combining  with  precipitins  and  forming  preci- 
pitates.    In  such  a  case  the  precipitinogen  can  be  employed  for  immunization 
purposes. 

A  constant  amount  of  precipitinogen  is  placed  into  each  of 
Technique  of  a  row  of  test  tubes,  and  to  these  are  added  diminishing  amounts 
the  Reaction,  of  the  serum. 

A  set  quantity  of  serum  and  varying  amounts  of  precipitinogen 
can  also  be  employed.  The  result  of  the  reaction  depends  to  a  very  large 
extent  upon  the  quantitative  relationship  of  these  ingredients.  If  relatively 
too  much  precipitinogen  exists,  a  precipitate  will  not  form.  An  already  formed 
precipitate  will  dissolve  on  the  addition  of  more  precipitinogen. 


no 


PRECIPITINS. 


The  explanation  of  this  peculiarity  is  unknown.  Since  colloidal  sub- 
stances, however,  at  times  give  similar  reactions,  many  authorities  have 
classed  the  precipitins  among  them. 

The  carrying  out  of  a  precipitation  test  is  best  seen  in  the  following  table: 


Cholera 

Cholera 

Physiological 

Result. 

bouillon 
filtrate. 

serum. 

saline  sol. 

After  4  hours. 

After  24  hours. 

5  .0  c.c. 

I.O      C.C. 



Very  cloudy.        Clear;  marked  sediment  at  bottom. 

5.0  c.c. 

0.5    c.c. 

0.5  c.c. 

Cloudy.           Clear    with    moderate    sediment    at 

bottom. 

5  .0  c.c. 

O.I      C.C. 

0.9    c.c. 

Faint  cloud.        Clear;  slight  sediment. 

5  .0  c.c. 

0.05  c.c. 

0.95  c.c. 

Clear. 

Clear;  no  sediment. 

5  .0  c.c. 

- 

I  .00  C.C. 

Clear. 

Clear;  no  sediment. 

— 

I.O      C.C. 

5.0    c.c. 

Clear. 

Clear;  no  sediment. 

— 

0.5    c.c. 

5-5    c.c. 

Clear. 

Clear;  no  sediment. 

- 

O.I      C.C. 

5.9   'c.c. 

Clear. 

Clear;  no  sediment. 

_ 

0.05  c.c. 

5.95  c.c. 

Clear. 

Clear;  no  sediment. 

A  parallel  row  of  tubes  with  normal  serum  should  be  included. 
If  highly  valent  sera,  such  as  are  obtained  by  immunization  with  bacterial 
extracts,  are  employed,  precipitation  may  result  soon  after  mixing  the  two 
constituents.  The  precipitins  are  strongly  specific,  although  it  may  be  said 
that  just  as  in  agglutination,  there  exists  in  precipitation  a  certain  degree  of 
"group  reactions" 

The   precipitation   test   has  no    clinical   diagnostic   -value.     It 

Diagnostic    demonstrates  nothing  more  than   the   agglutination   test,   is 

Value  of      more  difficult  of  execution  and  associated  with  greater  sources 

Bacterial      of   error.     Only   occasionally  is  it   of   service   to   prove   the 

Precipitation.  presence  of  soluble  bacterial  substances  within  exudates  or 

organ  fluids. 

Forges  and  v.  Eisler  have  employed  the  precipitation  test  as  a  means  for  the  differen- 
tiation of  capsule-bacteria  where  the  method  of  agglutination  is  associated  with  certain 
difficulties.  The  precipitinogen  was  produced  by  nitration  of  four-weeks-old  bouillon 
cultures  of  pneumococci,  rhinoscleroma,  and  ozoena  bacilli.  The  immune  serum  was 
obtained  from  rabbits  which  had  received  four  to  five  subcutaneous  injections  of  the 
bacterial  suspensions. 

Fornet  has  recently  advocated  the  precipitation  test  as  an  aid  in  the 
clinical  diagnosis  of  typhoid.  Although  his  attempts  have  not  been  attended 
with  practical  success,  the  principles  of  the  reaction  deserve  discussion  on 
account  of  their  originality. 

Fornet  believed  that  it  should  be  possible  to  demonstrate  in  the  blood  of  typhoid 
patients  the  presence  of  the  antigens  (precipitinogens)  which  stimulate  the  antibodies,  long 
before  the  latter  themselves  become  evident.  He  actuallv  was  able  to  obtain  turbid 


BACTERIAL    PRECIPITIN    REACTIONS.  Ill 

mixtures  when  he  combined  precipitating  typhoid  serum  with  the  serum  of  typhoid 
patients.  In  many  cases  he  obtained  these  results  before  the  appearance  of  the  Gruber- 
Widal  reaction. 

The  method  which  he  has  recently  employed  is  known  as  the  "ring  test." 

Small  test-tubes  8  cm.  in  height  and  0.5  cm.  wide,  are  placed  in  rows  of 
Fornet's       twenty  each  into  a  small  black  test-tube  rack  so  arranged  by  the  help  of 
Ring  Test,     side  stands  that  the  tubes  are  inclined  at  an  angle  of  about  45°.     Across 
the  back  of  the  rack  is  attached  a  strip  of  dark  cloth  as  a  background  to 
facilitate  the  detection  of  any  precipitate.     The  immune  (or  convalescent)  serum  is 
placed  into  different  tubes  in  concentrated  and  diluted  form  i  :  5  and  i  :  10  with  normal 
saline,  and  then  the  serum  for  examination  in  concentrated  and  similar  dilutions  is  care- 
fully floated  on  top  of  the  immune  serum.     The  mixtures  are  allowed  to  stand  undis- 
turbed at  room  temperature  for  two  hours,  and  if  the  reaction  is  positive  a  whitish  ring  at 
the  point  of  contact  of  the  two  sera,  makes  its  appearance.     A   control  test-tube  of 
normal  serum  plus  immune,  and  another  of  normal  plus  the  unknown  serum  in  the  same 
dilutions  as  those  employed  in  the  test,  must  remain  negative. 

Besides  in  typhoid,  the  ring  test  is  also  evident  in  scarlet  fever,  measles  and  syphilis. 

In  syphilis  precipitation,  the  serum  from  patients  with  mani- 
Syphilis       fest  luetic  symptoms  is  employed  as  precipitinogen,  and  the 
Precipitation,  serum  from  individuals  with  general  paresis  acts  as  precipitat- 
ing agent.     The  ring  test  must  be  carried  out  strictly  in  accord- 
ance with  the  rules  given  by  Fornet,  but  even  so,  its  diagnostic  value  for 
syphilis  is  still  doubtful.     Plaut  claims  that  normal  serum  gives  the  reaction 
just  as  often  as  luetic  serum;  this  is  strongly  denied  by  Fornet. 

Theoretically,  it  is  questionable  whether  these  precipitates  and  rings  are 
similar  in  origin  to  bacterial  precipitates,  or  whether  physicial-chemical 
causes  are  at  the  bottom  of  the  former  phenomenon.  In  accordance  with 
the  latter  view  several  other  reactions  have  been  recently  recommended 
for  the  serum  diagnosis  of  syphilis. 

a.  Forges  and  Meier  noticed  that  luetic  sera  are  capable  of  producing 
Forges'       flocculent  precipitates  from  lecithin  solutions.     Forges  soon  found  the 
Reaction.      same  occurrence  with  solutions  of  bile  salts. 

Many  additions  and  modifications  have  been  instituted  in  the  case  of 
Forges'  reaction  since  it  was  first  recommended.     According  to  the  most  recent  publica- 
tion, the  reaction  is  carried  out  as  follows: 
The  requirements  are: 

1.  One  per  cent,  solution  of  sodium  glycocholate  (Merck)  in  distilled  water. 

2.  The  patient's  serum  which  must  be  absolutely  clear,  and  heated  for  one-half  an 
hour  at  56°  C. 

Two-tenths  of  each  of  the  above  are  placed  into  a  narrow  test-tube  6  to  7  mm.  in  diam- 
eter, and  allowed  to  rest  for  sixteen  to  twenty  hours  at  room  temperature.  A  positive 
reaction  consists  of  the  appearance  of  distinct  coarse  flocculi  which  as  a  rule,  collect  near 
the  surface.  Mere  turbidity  or  faint  precipitates  are  considered  as  negative. 

The  original  Forges  method  of  employment  of  lecithin  was  not  at  all  specific,  the  reac- 
tion being  present  in  tuberculosis,  carcinoma,  and  other  infectious  diseases.  As  for  the 
new  modifications,  nothing  has  been  brought  forward  in  their  support  or  non-support. 


112  PRECIPITINS. 

This  reaction  belongs  to  the  same  general  class  of  precipitation 

Klausner's    tests  for  lues,  but  is  very  much  simpler  than  any  of  the  others. 

Reaction.     Two-tenths  c.c.  of  absolutely  clear,  fresh  (at  the  most,  two 

hours  old),  active  serum  is  mixed  with  0.6  c.c.  of  distilled  water, 

in  a  small  test-tube  7  X  0.5  cm.     Sera  containing  hemoglobin  or  lipoids 

are  not  suitable  for  this  reaction.     The  mixtures  are  allowed  to  stand  at 

room  temperature.     In  several  hours,  at  the  latest  fifteen,  a  thick  flocculent 

precipitate  2  to  4  mm.  high  appears  at  the  bottom  of  the  tube.     Kreibich's 

analysis  showed  it  to  consist  of  fibrin  globulin. 

Apparently  this  substance  is  increased  in  luetic  serum  and  precipitated  by  the  dis- 
tilled water  in  which  it  is  insoluble.  Klausner's  reaction  is  by  no  means  specific  for 
syphilis  as  it  is  in  evidence  in  starvation,  typhoid,  measles,  scarlet,  pneumonia,  and  other 
diseases,  as  well  as  during  health.  Nevertheless  it  must  be  said  that  it  is  found  more 
frequently,  earlier  and  much  stronger  in  lues  than  in  any  other  condition. 

Klausner  states  that  in  fresh  cases  of  lues  the  best  reaction  is  seen  in  about  seven  to 
nine  hours,  while  in  older  cases  a  week  reaction  appears  in  twelve  hours.  Mercury 
influences  the  test  in  that  the  interval  until  the  precipitate  becomes  marked,  is  prolonged 
and  later  on  the  precipitate  becomes  fainter. 

In  spite  of  its  simplicity,  Klausner's  reaction  has  not  been  generally  adopted  for 
clinical  work,  inasmuch  as  the  far  greater  accuracy  of  the  Wassermann  reaction  has  made 
the  latter  invaluable. 


Proteid  Precipitins. 

While  bacterial  precipitation  is  interesting  from  a  biological  standpoint 
but  bears  no  practical  significance,  proteid  precipitation  represents  one  of 
the  most  important  practical  aids  in  forensic  medicine.  By  this  means  the 
differentiation  of  various  proteids  can  be  easily  and  definitely  determined,  a 
problem  which  was  left  unsolved  by  chemistry. 

The  phenomenon  of  protein  precipitation  is  absolutely  analogous  to 
that  of  bacterial  precipitation.  If  a  clear  proteid  solution  (a)  is  mixed  with 
the  clear  serum  (a')  of  an  animal  immunized  against  the  above  proteid  (a) , 
turbidity  and  precipitation  will  occur;  while  if  a  mixture  of  the  serum  (a')  is 
made  with  a  non-homologous  proteid  say  (b) ,  or  a  mixture  of  the  proteid  (a) 
with  the  serum  (bf)  of  an  animal  immunized  against  b,  no  precipitation 
takes  place.  Graphically  expressed  it  looks  thus: — 

a  +  a!  =  precipitation. 
b  +  a'  =no  precipitation. 
a  +  bf  =no  precipitation. 
b+b'  =  precipitation. 

In  other  words,  a  precipitating  immune  serum  reacts  only  with  its  homol- 
ogous proteid.  The  precipitin  reaction  is  specific. 


PROTEID    PRECIPITINS.  113 

It  is  greatly  to  the  credit  of  Wassermann  and  his  co-workers 
Forensic  Use  A.  Schutze  and  Uhlenhuth,  who  recognized  that  this  speci- 
of  Albumin    ficity  of  precipitins  was  of  great  medico- legal  value. 
Differentia- 

tion  Thus  in  a  case  where  for  example,  a  bloody  shirt  is  found  in  the  home 

of  a  man  charged  with  murder,  and  the  prosecution  sees  in  that  the 

proof  of  crime,  while  the  defendant  pleads  that  the  stains  belong  to  the 

blood  of  a  sheep,  the  proof  as  to  their  source  is  of  the  utmost  deciding  evidence;  and 

while   chemical   or   microscopical   examinations   are    here  of   little   or  no  use,  serum 

diagnosis  wins  the  day. 

The  blood-stained  clothing  is  extracted  in  water,  part  of  the  extract  is  mixed  with  a, 
the  serum  of  a  rabbit  immunized  against  human  serum  and  another  part  is  mixed  with  b, 
the  serum  of  a  rabbit  immunized  against  sheep's  serum.  If  the  mixture  a  shows  a  pre- 
cipitate, it  can  be  definitely  stated  that  the  blood  stain  contained  serum  derived  from  a 
human  being;  while  if  mixture  a  is  clear  and  b  shows  the  precipitate,  it  is  strongly  corrob- 
orative of  the  presence  of  sheep's  serum. 

This  example  suffices  to  indicate  the  value  of  this  biological 
Blood        fact.     In  addition  the  reaction  is  made  use  of  in  the  deter- 
Relationship.  mination  of  the  nature  of  meats  (detection  of  horse  meat  sub- 
stitution for  beef). 

Furthermore,  this  method  has  explained  a  number  of  scientifically  inter- 
esting problems.  Just  as  group  agglutination  demonstrated  the  close 
relationship  existant  between  various  bacteria,  so  also  serum  precipitation 
proves  a  distinct  relationship  between  the  different  species  of  animals 
(horse  and  donkey,  dog  and  fox,  hare  and  rabbit,  ape  and  man,  etc.). 

Thus  the  serum  of  a  rabbit  immunized  against  human  serum,  precipitates  not  only 
human  serum  but  also  that  of  monkeys;  the  serum  of  a  chicken  immunized  against  rabbit's 
serum  precipitates  not  only  that,  but  also  hare's  serum.  In  order,  however,  to  differentiate 
between  rabbit's  and  hare's  serum,  Uhlenhuth  advises  the  immunization  of  a  rabbit  with 
hare's  serum.  The  serum  of  such  an  immunized  rabbit,  precipitates  only  hare's  serum 
and  not  rabbit's,  for  the  reason  that  " Isoprecipitins, "  i.e.,  precipitins  against  the  same 
kind  of  animal,  are,  as  a  general  rule  not  developed.  Similarly  the  differentiation 
between  human  and  ape's  serum  can  be  accomplished  by  the  immunization  of  ape's 
with  human  serum. 

Attempts  to  determine  the  origin  of  albumin  in  urine,  and  the  foreign 
proteids  circulating  in  the  blood  of  artificially  fed  infants  have  also  been 
made  by  means  of  the  precipitation  reaction. 

The  technique  remains  the  same,  independent  of  the  purpose  it  is  em- 
ployed for.  It  consists  in  the  mixing  of  the  clear  precipitating  serum  and 
the  clear  proteid,  or  albumin  precipitinogen. 

In  order  to  obtain  accurate  results,  strongly  precipitating  sera  must  be  had.  These 
are  best  made  by  immunizing  rabbits  with  the  precipitinogen  fluid  (albumin  solution, 
milk,  meat  juice,  etc.).  Three  to  four  intravenous  injections  with  i  c.c.  of  the  solution 
at  intervals  of  six  days  usually  suffice  to  produce  a  precipitating  serum  of  high  titer. 
The  injections  can  also  be  given  by  the  intramuscular  or  subcutaneous  method,  but  here 
larger  quantities  are  necessary. 
8 


114  PRECIPITINS. 

It  is  advisable  to  inject  five  or  six  animals  at  the  same  time,  instead  of  only  one,  inas- 
much as  rabbits  vary  greatly  in  their  individual  power  to  produce  precipitins  and  more- 
over, because  some  die  after  the  third  injection.  Frequently  only  one  serviceable  serum 
is  obtained,  even  though  the  immunization  of  five  rabbits  was  undertaken. 

Beginning  on  the  sixth  day  after  the  injection,  one  should,  at  regular 
intervals  of  one  or  two  days,  remove  a  small  quantity  of  blood  from  the  vein  of 
an  ear  and  test  the  strength  of  the  serum.  As  soon  as  it  is  found  to  be 
satisfactory  the  animal  should  be  bled  and  its  serum  preserved  on  ice,  with 
precautions  for  sterility.  The  rules  given  above  for  obtaining  a  clear  serum 
should  be  kept  in  mind. 

If  the  serum  is  not  withdrawn  at  the  proper  time,  its  strength  begins  to  diminish  and 
further  injections  no  longer  stimulate  new  antibodies.  It  is  even  possible  for  the  entire 
precipitin  action  of  the  serum  to  disappear. 

The  following  method  of  titration  is  the  simplest.     One  c.c. 
Titration. 

of  various  dilutions  (i  :io,  i :  100,  i :  1000,  i :  10000)  of  the  pro- 

teid  under  examination  (precipitinogen)  is  placed  into  different  test-tubes 
and  o.i  c.c.  of  the  precipitating  serum  is  added  to  each.  The  tubes  should 
not  be  shaken,  but  it  is  occasionally  necessary  to  place  them  into  the  incu- 
bator for  one  hour  before  any  turbidity  or  precipitate  appears.  The  least 
amount  of  proteid  solution  which  still  distinctly  shows  a  precipitate,  is 
taken  as  the  titer  of  the  serum. 

For  medico-legal  purposes,  Uhlenhuth  advises  the  use  of  only 

Uhlenhuth's    highl7  valent  sera- 

Method  of    He  considers  an  antiserum  as  efficient  if  o.i  c.c.  of  it,  when 
Proteid  Dif-  mixed  with  its  respective  serum  in  the  dilution  of  1:1000, 
ferentiation.   produces  a  distinct  turbidity,  either  at  once  or  one  to  two  min- 
utes at  the  latest;   three   to  five  minutes  is  the  limit  for  an 
indication  of  turbidity  in  the  dilutions  of  i :  10000  and  i :  20000. 

Like  in  all  other  biological  reactions,  control  tests,  here  two  in  number, 
are  of  the  utmost  importance.  One  tube  must  contain  o.i  c.c.  of  the  precipi- 
tating serum  mixed  with  i  c.c.  of  saline,  another  o.i  c.c.  of  the  precipitating 
serum  mixed  with  a  heterologous  serum  in  the  dilution  of  i :  200  and  i :  1000. 
Both  of  these  tubes  should  show  absolutely  no  precipitate  after  twenty 
minutes.  In  this  way  the  specificity  of  the  precipitin  is  determined; 
and  it  must  be  remembered  that  it  is  the  quantitative  specificity  which 
counts. 

In  the  process  of  the  determination  of  the  nature  of  meats,  it  is  especially 
necessary  to  ascertain  exactly  the  precipitating  titer  against  bovine  and  pig's 
serum  possessed  by  the  rabbit's  precipitating  serum  directed  against  horse's 
serum. 

When  clear  solutions  are  at  hand  the  precipitin  reaction  is  comparatively 
simple.  Frequently,  however,  the  test  must  be  performed  with  old  and  dirty 


PROTEID    PRECIPITINS. 

blood  stains,  or  all  kinds  of  prepared  sausage  so  that  the  first  and  important 
task  is  to  obtain  a  clear  solution. 

In  dealing  with  blood,  milk,  or  seminal  stains,  the  parts  of  the  clothing  involved  are 
excised,  divided  into  very  minute  shreds,  and  placed  into  a  test-tube  with  a  small  amount 
of  0.85  per  cent,  of  salt  solution.  If  the  material  is  not  too  old,  extraction  of  the  above 
nature,  for  one  hour  is  usually  sufficient,  otherwise  it  may  necessitate  a  period  of  twenty- 
four  hours  or  more.  Stains  upon  solid  material  such  as  steel,  wood,  stone,  etc.,  are  care- 
fully scraped  off,  and  suspended  in  physiological  salt  solution.  To  obtain  a  clear  solution 
the  extract  must  be  passed  through  filter  paper  or  eventually  the  lilliputian  bacterial 
filter. 

In  the  examination  of  meats,  or  other  food  stuffs,  it  is  best  to  remove  the 
material  for  examination  from  the  center  of  its  thickest  part,  as  this  portion 
has  been  least  exposed  to  the  methods  of  preservation,  especially  the  high 
temperatures.  Three  hours  extraction  is  usually  sufficient;  the  fresher  the 
meat.,  the  shorter  this  period.  Very  much  salted  meats  are  best  washed 
with  distilled  water,  previous  to  extraction.  Inasmuch  as  a  great  deal  of 
fat  interferes  with  the  reaction  it  is  advisable  to  remove  it  beforehand  by 
extraction  wth  ether  and  chloroform  for  twenty-four  hours  (Miessner 
and  Herbst). 

Before  performing  the  actual  test  with  the  unknown  blood  stain,  it  is 
best  to  try  out  the  entire  reaction  with  a  similar  but  known  blood  stain  in 
order  to  make  sure  whether  all  the  ingredients  are  in  good  working  order. 
In  laboratories  equipped  for  medico-legal  examinations,  stains  made  upon 
linens  from  the  blood  of  man,  ox,  pig,  horse,  etc.,  are  always  kept  in  readiness 
for  such  preliminary  tests. 

Uhlenhuth  indicates  a  set  of  rules  to  be  observed  whenever  the  reaction 
is  undertaken.  They  are  here  cited  in  their  original  form,  as  practice  has 
shown  them  to  be  of  great  service. 

"In  order  to  obtain  sufficient  extract  for  the  test,  a  small  amount  of  the  material  is 
placed  into  a  test-tube  containing  5  c.c.  of  normal  salt  solution.  This  must  not  be  shaken. 
After  one  to  two  hours,  2  c.c.  are  poured  off  into  another  tube  and  gently  shaken.  If  a 
persisting  froth  appears  upon  the  surface  of  the  fluid,  it  can  be  taken  as  proof  that  suffi- 
cient extraction  has  occurred,  and  the  rest  of  the  fluid  is  thereupon  also  transferred  to 
this  tube.  If  no  froth  appears  the  2  c.c.  should  be  returned  into  the  first  test-tube  and 
the  extraction  continued  until  repeated  tests  finally  show  the  presence  of  froth.  It  is 
preferable  not  to  disturb  the  sediment  at  the  bottom  of  the  test-tube.  The  extract  even- 
tually obtained  may  have  to  be  filtered,  if  not  absolutely  clear. 

Such  an  extract  is  as  a  rule,  stronger  than  that  required  for  the  test,  i.e.,  i  :  1000.  If, 
however,  one  drop  of  a  25  per  cent,  nitric  acid  solution  is  added  to  i  c.c.  of  a  i  :  1000 
serum  dilution  and  then  heated,  a  faint  opalescence  appears.  Enough  saline  should 
therefore  be  added  to  the  final  extract  so  that  the  nitric  acid  test  corresponds  to  that 
given  by  a  dilution  of  i  :  1000. 

The  following  mixtures  are  then  made: 


n6 


PRECIPITINS. 


Precipitating 

Normal 

Result 

Test  solution. 

serum  from 
rabbit. 

rabbit's 
serum 

saline. 

After  five 

After  twenty 

minutes. 

minutes. 

i  c.c.   1:1000 

O.  I 





Opalescence. 

Turbidity  and  sedi- 

ment. 

i  c.c.  1:1000 

— 

O.I 

— 

Clear. 

Clear. 

O.  I 

~~  " 

I  C.C. 

Clear. 

Clear. 

The  result  should  be  read  after  twenty  minutes  at  room  temperature. 
As  a  further  control  a  similar  row  of  tubes  should  be  made  with  the  extract 
of  the  non-bloody  part  of  the  clothing  in  order  to  show  that  the  latter  alone 
does  not  give  the  reaction. 

Even  putrid  or  otherwise  chemically  changed  proteids  may  still  give  the 
precipitin  reaction. 

The  precipitation  test  only  determines  the  animal  species  from  which 
the  proteid  originates,  but  cannot  prove  whether  it  comes  from  the  blood, 
semen,  milk  or  other  albumin  body.  In  order  therefore  to  make  a  medico- 
legal  diagnosis  of  "  human  blood  stains,"  chemical  evidences 
" Origin"  and  must  *n  addition  be  brought  forward,  that  the  stain  really 
"Constitu-  consists  of  blood.  Obermeyer  and  Pick  have  further  shown 
tional"  Spec-  that  besides  animal  specificity  ("origin  specificity"),  pre- 

ificity.  cipitation  also  demonstrates  the  "constitutional  specificity"  of 
proteids. 

If  instead  of  employing  pure  animal,  or  plant  proteids  for  the  immu- 
nization of  animals,  variously  changed  albumins  are  used  (heated  albumins, 
acid  albumins,  formaldehyde  albumin,  etc.)  the  organism  reacts  by  produc- 
ing antibodies  of  a  characteristic  nature,  different  from  those  developed 
after  inoculation  with  the  pure  albumin.  For  example;  the  serum  of  a 
rabbit  immunized  for  a  long  time  with  horse's  serum  (normal  immune  pre- 
cipitin) will  produce  a  precipitate  when  mixed  in  vitro  with  the  pure  horse's 
serum  and  not  when  added  to  the  latter,  heated,  even  if  the  normal  immune 
serum  is  of  very  high  titer.  On  the  other  hand,  if  a  rabbit  is  injected  with 
horse's  serum  which  has  been  changed  by  being  diluted  and  boiled  for  a 
short  time,  the  immune  serum  thus  obtained  will  react  not  only  with  native 
horse's  serum  but  also  with  heated  serum  and  a  group  of  its  decomposition 
products  with  which  the  normal  immune  serum  ordinarily  never  induces  a 
precipitate. 

This  fact  is  of  practical  application.  In  meat  substitution,  it  is  very  popular  to  boil 
the  sausage  in  order  to  make  detection  of  the  substituted  meats  more  difficult.  With  the 
aid,  however,  of  precipitins  produced  by  immunization  with  heated  proteids,  this  fabrica- 
tion is  more  easily  detected  than  if  a  normal  immune  serum  were  used. 


ORIGIN    OF    PRECIPITATE. 


117 


While  animal  specificity  is  not  destroyed  when  the  albumins 
Precipitin.       are  modified  in  the  above  manner  or  changed  by  tryptic  diges- 
tion or  oxidation,  Obermeyer  and  Pick  have  demonstrated  that 
their   specificity  is  lost  when  an  iodin,  nitro  or  diazo  group  is   inserted 
into   the  proteid  molecule.     Immunization  with  such  transformed  proteid 
compounds,  e.g.,  xanthoprotein,  can  produce  a  precipitating  serum  which 
will  react  with  every  xanthoprotein  even  in  homologous  animals.     These 
authors   conclude  that  species  specificity  is  probably  dependent  upon   a 
certain  aromatic  group  of  the  proteid  molecule. 

It  is  interesting  to  note  that  the  proteid  contained  in  the  lens  of  the  eye 
belongs  to  this  class  of  modified  proteids  which  possess  constitutional, 
but  no  species  specificity.  A  serum  produced  by  immunization  with  lens 
substance,  will  react  with  the  proteid  derived  from  the  lens  of  any  animal 
but  with  no  other  animal  proteid. 

In  conclusion,  the  origin  of  the  precipitate  formed  during  the 
Origin  of     precipitation    reaction  is  of  interest.     When  a  very  strong 
Precipitate,    precipitating   serum   is   employed,   the   precipitinogen  is   so 
greatly  diluted  that  it  no  longer  gives  any  of  the  chemical 
reactions  for  proteids,  but  nevertheless  yields  a  heavy  precipitate  when  the 
precipitating  serum  is  added.     This  surely  cannot  come  from  the  small 
trace  of  proteid  in  the  precipitinogen.     Furthermore,  if  the  immune  serum 
is  diluted,  the  formed  precipitate  becomes  comparatively  weaker  and  dis- 
appears entirely  if  dilution  is  increased.     It  is,  therefore,  generally  considered 
that  the  precipitate  originates  from  the  immune  serum. 


CHAPTER  XII. 
BACTERIOLYSINS  AND  HEMOLYSINS  (CYTOLYSINS). 

If  a  guinea-pig  is  immunized  with  living  or  dead  bacteria,  for  instance 
cholera  or  typhoid,  and  then  to  test  its  immunity  is  injected  with  a  single 
fatal  or  many  fatal  doses  of  living  bacilli,  the  animal  remains  alive;  whereas 
a  normal-control  animal,  not  treated  beforehand,  succumbs  to  a  similar 
inoculation.  In  order  to  determine  the  forces  to  which  the  immunized 
animal  owes  its  protection,  Pfeiffer  undertook  the  following  experiment: 
Two  guinea-pigs,  one  immunized  and  another  normal,  were  simultaneously 
injected  intra-peritoneally  with  living  cholera  vibrios,  and  the  peritoneal 
exudate  was  withdrawn  from  time  to  time  and  examined  microscopically 
in  hanging-drop  preparations.  (The  method  of  withdrawing  the  peritoneal 
fluid  with  capillary  pipettes  and  other  technical  details  will  be  described 
below.) 

A  very   striking   phenomenon   occurred.    While   the   cholera 

Pfeiffer's      vibrios  in  the  peritoneal  exudate  of  the  normal  animal  retained 
Phenomenon,  their  form  and  motility  and  increased  in  number  continuously 
until  the  animal  succumbed  to  the  infection,  the  bacteria  in 
the  peritoneal  exudate  of  the  immunized  animal  behaved  quite  differently; 
they  first  began  to  lose  their  power  of  locomotion,  then  their  form  changed, 
they  broke   up    into    evenly  small   shining  masses,  so-called  "granula," 
and  finally,  after  several  minutes  these  also  disappeared.     Guinea-pigs  in- 
jected with  the  peritoneal  exudate  from  these  infected  immune  animals 
remained  healthy,  and  nutrient  media  inoculated  with  material  from  the 
same  source  remained  sterile. 

The  above  experiment  is  named  after  its  discoverer,  Pfeiffer,  and  the 
phenomenon  itself,  "bacteriolysis." 

Bacteriolysis  is  a  strictly  specific  process.  If  an  animal  which  is  immune 
to  cholera  is  inoculated  with  typhoid  bacilli,  the  bacteria  markedly  increase, 
as  in  a  normal  animal.  The  process  by  which  this  bacteriolytic  force  takes 
place  is  clearly  demonstrated  when  a  mixture  of  living  cholera  vibrios  and 
blood  serum  of  a  guinea-pig  which  has  been  actively  immunized  against 
cholera,  is  injected  into  the  peritoneal  cavity  of  a  normal  guinea-pig  and  as  a 
control,  normal  serum  mixed  with  living  cholera  vibrios  is. inoculated  into  a 
second  guinea-pig.  Here  the  exudates  on  examination  from  time  to  time 
show  that  in  the  peritoneal  cavity  of  the  animal  injected  with  the  immune 

118 


PFEIFFER  S    PHENOMENON.  Up 

cholera  serum,  the  same  phenomena  of  bacteriolysis  occur  as  described 
above,  leading  to  the  sterilization  of  the  peritoneal  cavity,  and  protection  of 
the  animal  from  illness.  In  the  control  animal,  however,  the  normal  serum 
has  no  influence  upon  the  bacteria,  so  that  they  increase  rapidly  and  kill 
the  animal. 

It  is  evident  then,  that  the  bacteriolytic  power  resides  not  only  in  the 
actively  immunized  animal,  but  that  it  may  also  be  transmitted  to  other 
animals  by  means  of  the  former's  serum.  Bacteriolysis,  therefore,  is  not  a 
property  of  the  tissues  of  the  actively  immunized  animal,  but  is  to  be  traced 
to  specific  antibodies,  "Bacteriolysins"  which  circulate  in  the  blood  serum 
and  body  fluids. 

From  the  above  experiment  it  must  be  assumed  that  the  phenomenon  of 
bacteriolysis  like  agglutination  and  precipitation,  can  be  demonstrated  also 
in  vitro.  The  earlier  investigations  in  this  connection,  however,  were 
unsuccessful.  Bordet  was  the  first  to  obtain  conclusive  results  and  also  to 
elucidate  the  cause  of  previous  failures. 

While  agglutination  in  vitro  and  bacteriolysis  in  vivo  were  readily  pro- 
duced by  mixing  living  bacteria  with  old  immune  serum,  bacteriolysis  in 
vitro  did  not  occur  under  similar  circumstances.  But  when  freshly  drawn 
blood  serum  or  exudate  of  an  immune  animal  was  used,  bacteriolysis  took 
place  in  vitro  also.  (In  fact,  granule  formation  can  be  directly  observed  by 
the  microscope).  When  the  serum  becomes  old — and  twenty-four  hours  is 
sufficient  to  cause  the  change,  it  loses  its  bacteriolytic  powers.  It  seems  at 
first  glance  as  if  bacteriolysins  may  be  active  outside  the  body  also,  but  that 
here  they  lead  only  an  ephemeral  existence.  This  view,  however,  is  not 
quite  correct;  for  "inactive"  serum,  which  has  become  "ineffective"  in 
vitro,  can  again  produce  bacteriolysis,  if  it  is  utilized  to  passively  immunize 
healthy  animals.  Something  must  exist  in  the  organism,  which  supplements 
the  inactive  bacteriolysins  and  restores  their  activity.  This  "reactivating 
substance"  is  independent  of  the  immunizing  process,  since  it  is  to  be  found 
in  normal  animals  also.  Furthermore,  inasmuch  as  not  only  cholera  and 
typhoid  immune  sera,  but  also  any  other  immune  sera  and  not  only  guinea- 
pig's  serum  but  even  rabbit's,  horse's,  and  human  serum  may  in  like  manner 
be  reactivated,  it  is  evident  that  the  reactivating  agent  lacks  specificity.  On 
account  of  this  peculiar  quality  of  supplementing  the  inactive  bacteriolytic 
serum  so  that  it  can  develop  its  real  effectiveness,  Ehrlich  called  the  reactivat- 
ing substance  "Complement"  Accordingly,  the  complement  is  a  normal 
non-specific  substance  which  is  found  in  the  body  fluids  (particularly  abundant 
in  the  blood  serum)  of  every  organism;  its  existence  is  evidenced  either  by  the 
activation  or  reactivation  of  bacteriolytic  antibodies. 

Bordet  demonstrated  that  the  apparent  ease  with  which  the  bacteriolysins 
lose  their  activity  is  to  be  traced  not  to  these  bodies,  but  to  the  complement. 
If  a  small  amount  of  fresh  normal  serum  is  added  to  bacteriolytic  serum 


120  BACTERIOLYSINS   AND   HEMOLYSINS. 

which  has  become  inactive,  reactivation  occurs  in  vitro,  that  is  to  say,  the 
bacteriolytic  serum,  regains  its  ability  to  dissolve  bacteria.  The  bacterio- 
lytic  power  of  fresh  immune  serum,  depends,  therefore,  upon  the  fact  that  it 
contains  not  only  bacteriolysins  but  also  complement;  the  failure  of  old  im- 
mune serum  to  produce  bacteriolysis  is  accounted  for  by  the  lack  of  comple- 
ment, while  its  capacity  for  reactivation  is  explained  by  the  still  present 
bacteriolysins. 

As  the  above  described  experiments  indicate,  bacteriolysis  is  a  complex 
process,  which  is  produced  by  the  interaction  of  two  substances;  one,  the  bac- 
teriolysin, is  formed  through  an  immunizing  process,  and  accordingly  is  a 
specific  antibody  of  great  stability,  while  the  other,  the  complement,  is  a 
normal  non-specific  and  very  labile  serum  substance. 

The  stability  of  the  immune  bacteriolysin  is  evident  in  its  resistance  to 
heat,  whereas  the  complement  is  thermolabile.  If  freshly  drawn  immune 
serum  is  heated  to  56°  C.  for  one-half  hour,  the  complement  is,  as  a 
rule,  rendered  ineffective,  while  the  -bacteriolysin  is  not  in  any  way  in- 
jured; it  retains  its  specificity,  and  the  degree  of  its  affinity  to  antigen 
remains  unchanged.  Bacteriolysins  are  effected  by  temperatures  above 
60°  C.  only. 

Coficerning  the  finer  mechanism  of  bacteriolysis  there  are  two  opposing  views,  that 
of  Bordet  and  of  Ehrlich.  Without  considering  too  closely  the  remarkable  researches  of 
these  two  investigators,  the  synonyms  for  bacteriolytic  antibodies  usually  found  in  the 
literature  will  be  reviewed. 

In  attempting  an  explanation  of  bacteriolysis,  Bordet  has  recourse  to  certain  phenom- 
ena in  staining  technique.  There  are  some  substances  which  can  be  stained  only  when 
prepared  in  a  definite  way  by  means  of  another  substance,  a  so-called  mordant  ("Beize") 
which  itself  is  not  a  stain.  According  to  Bordet,  the  specific  substance  produced  by 
immunization  represents  a  kind  of  mordant  which  "sensitizes"  the  bacteria  to  the  action 
of  the  second  normal  non-specific  substance;  the  latter  is  really  the  active  agent  in  causing 
the  dissolution  of  bacteria  and  is  called  by  Bordet  "alexin" — an  older  term  used  by 
Buchner — in  contradistinction  to  " substance  sensibilitrice." 

Ehrlich,  on  the  other  hand,  advocates  a  more  chemical  conception  of  the  essential^ 
process  of  bacteriolysis.  He  believes  that  the  substance  formed  by  immunization  which 
for  the  sake  of  brevity,  is  called  the  immune  body,  is  characterized  primarily  by  the  fact 
that  it  has  two  binding  groups.  One  of  these  has  a  chemical  affinity  for  the  bacterial  cell 
and  is,  therefore,  known  as  the  "cytophile  group,"  the  other  is  characterized  by  its  binding 
affinity  for  complement  and  is,  therefore,  known  as  the  "complementophile"  group. 
Also  because  of  its  two  binding  groups  (receptors)  the  immune  body  itself  is  called  ambo- 
ceptor,  that  is,  double  receptor. 

Thus,  according  to  Ehrlich,  bacteriolysis  takes  place  in  the  following  way:  The  cyto- 
phile group  of  the  amboceptor,  which  is  strictly  specific  for  its  antigen,  attaches  itself  to 
the  antigen,  for  instance  the  cholera  vibrio;  while  the  complementophile  group  binds  the 
complement.  The  complement  must  be  regarded  as  a  sort  of  digesting  (proteolytic) 
ferment.  Although  it  is  always  present  in  normal  serum,  it  is  not  effective,  because 
bacteria  have  no  affinity  for  it.  Only  through  the  medium  of  the  amboceptor,  (Zwischen- 
Korper,  intermediary  body),  can  complement  bind  itself  to  bacteria  and  dissolve  them. 


TECHNIQUE    OF    BACTERIOLYTIC    EXPERIMENTS. 


121 


The  specificity  of  the  bacteriolytic  process  depends,  therefore,  on  the 
specificity  of  the  cytophile  group,  while  the  complementophile  group  pos- 
sesses no  or,  strictly  speaking,  only  slight  specificity;  it  adapts  itself  to  the 
complements  of  very  many  though  not  quite  all  kinds  of  animals. 

Technique  of  Bacteriolytic  Experiments. 

To  determine  the  occurrence  of  bacteriolysis  there  are  two  methods  of 
procedure; 

1.  Pfeiffer's  experiment. 

2.  The  bactericidal  plate  method. 


I.  The  Pfeiffer's  Experiment. 

The  essentials  of  Pfeiffer's  experiment  have  been  described  at  the  begin- 
ning of  this  chapter.  Briefly,  it  consists  in  injecting  intraperitoneally  into  a 
normal  animal,  bacteriolytic  immune  serum  mixed  with  living  bacteria. 
The  resulting  bacteriolysis  is  studied  microscopically  by  withdrawing  small 
amounts  of  peritoneal  exudate  from  time  to  time.  If  this  experiment  is 
performed  with  various  dilutions  of  immune  serum,  and  if  it  be  determined 
at  what  dilution  bacteriolysis  fails  to  occur,  then  the  bacteriolytic  titer  is 
evident. 

The  details  can  best  be  understood  by  taking  a  practical  example.  It  is 
desired  to  find  the  bacteriolytic  titer  of  the  serum  of  a  patient  recovering 
from  typhoid  fever  by  means  of  the  Pfeiff er  experiment. 

To  accomplish  this  task  the  following  ingredients  are  needed: 

1.  A  strain  of  bacillus  typhosus  of  known  virulence  for  guinea-pigs. 

2.  Patient's  serum,  sterile,  and  free  from  complement. 

3.  Guinea-pigs  of  250  grams  weight. 

A  preliminary  experiment  must  be  performed  in  order  to  determine  the  virulence  of  the 
typhoid  strain. 


TESTING  THE  VIRULENCE  OF  STRAIN. 


Guinea-pig  No.    i. 

i./  II.  09  One  loopful  of  a  typhoid   agar  culture  suspended  in  i   c.c 
of  bouillon,  injected   intraperitoneally  

2/II  dead 

Guinea-pig  No.    2. 

i  /II    09  One  half  loopful  of  same 

2/II  dead 

Guinea-pig  No     3 

i  /II    09  One-  fifth  loopful  of  same 

2/II  dead 

Guinea-pig  No.   4. 

i./II.  09  One-eighth  loopful  of  sams  

2/II  sick 

4/II  dead 

Guinea-pig  No.   5. 

i./II.  09  One-tenth  loopful  of  same  

2/II  Sick 

3/II  well 

122  BACTERIOLYSINS   AND   HEMOLYSINS. 

As  far  as  the  Pfeiffer  experiment  is  concerned  the  virulence  titer  in  this  case  is  1/5  of  a 
loopful  of  an  agar  culture  because  this  dose  is  fatal  within  twenty-four  hours.  In  order, 
however,  to  make  sure  of  excluding  all  individual  variations,  which  can  and  occasionally 
do  occur,  it  is  advisable  to  use  not  the  titer  dose,  but  its  fifth  or  tenth  multiple,  that  is,  in 
this  case,  one  loopful. 

Doses  larger  than  one  loopful  should  be  avoided,  so  that  if  any  particular 
strain  of  typhoid  bacilli  is  not  sufficiently  virulent,  necessitating  the  use  of 
larger  doses,  the  virulence  must  first  of  all  be  increased.  This  is  done  by 
passing  the  organism  through  animals  such  as  guinea-pigs. 

The  method  is  as  follows:  A  very  large  dose  of  the  culture,  for  example 

To  Increase    the  surface  of  an  entire  agar  tube,  is  injected  intraperitoneally.     Every 

the  Virulence,  animal  succumbs  to  this  enormous  dose.     The  bacteria-laden  exudate 

from  the  abdominal  cavity,  which,  of  course,  must  be  removed  under 
sterile  precautions  is  then  inoculated  into  a  second  guinea-pig  and  when  it  dies,  into  a 
third,  and  so  on.  As  a  rule,  after  passing  through  one  or  two  animals  the  bacterial  strain 
(which  must  be  grown  pure  from  the  cadaver)  becomes  more  virulent,  as  can  be  proven 
by  titration.  Very  often  the  virulence  is  increased  exclusively  for  the  species  of  animal 
used  and  occasionally  this  is  associated  with  a  decrease  in  virulence  for  other  species. 
After  a  series  of  passages  through  animals,  the  strain  reaches  its  maximum  strength 
beyond  which  it  cannot  be  increased.  The  degree  of  virulence  varies  with  the  type  of 
bacteria.  Typhoid  and  cholera  usually  reach  only  a  moderate  virulence  (i/io  to  1/20 
loopful);  the  bacteria  of  the  hog  cholera  group  can  acquire  a  distinctly  higher  virulence; 
for  instance,  B.  paratyphosus,  i/ioo  to  i/iooo  of  a  loopful,  while  the  streptococcus  and 
pneumococcus  reach  the  highest  figures,  i/ioooo  to  i/ioooooo  of  a  loopful. 

For  the  Pfeiffer's  experiment  with  cholera  or  typhoid,  the  most  suitable  strains  are 
those  of  such  a  virulence  that  1/5  to  i/io  of  a  loopful  injected  intraperitoneally  kills  in 
twenty-four  hours. 

The  serum  to  be  investigated  is  freed  of  its  serum  by  heating  in 

Technique  of  a  water-bath  for  one-half  hour  at  56°  C.     Then  a  series  of 

Pfeiffer's      dilutions  are  made  in  bouillon  (not  in  salt  solution)  for  instance 

Experiments,  i/io,  i/ioo,  i/iooo,  etc.    A  c.c.  of  each  dilution  is  put  into  a 

test-tube  (a  sterile  pipette  should  be  used)  and  rubbed  up  with 

a  standard  loopful  of  an  18-  to  24-hour  agar  culture  of  typhoid  bacteria. 

Finally  the  contents  of  each  test-tube  are  injected  intraperitoneally  into  a 

guinea-pig  of  250  grams  weight. 

Inasmuch  as  small  amounts  are  apt  to  be  lost  when  aspirating  the  fluid 
with  the  syringe  as  well  as  when  pouring  the  bacterial  emulsion  into  a  watch 
glass,  it  is  better  to  rub  up  two  loops  of  the  culture  in  2  c.c.  of  bouillon 
instead  of  i  loop  in  i  c.c.,  and  then  withdraw  only  i  c.c.  for  use  in  the 
experiment. 

The  following  controls  should  be  prepared: 

i.  Dilutions  of  the  serum  of  a  normal  person  (or  animal  of  the  same 
type)  f  typhoid  culture. 


STUDY    OF    BACTERIOLYTIC    PHENOMENA. 


I23 


2.  Dilutions  of  immune  serum  +  a  heterologous  culture. 

3.  (a)  Bouillon  +  typhoid-culture. 

(b)  Bouillon  +  heterologous  culture. 

The  study  of  the  bacteriolytic  phenomena  follows  the  inoculation.  For 
this  purpose  capillary  pipettes  to  withdraw  the  peritoneal  exudate  are  pre- 
pared according  to  the  directions  of  von  Issaeff. 

A  thin  glass  tube  is  heated  in  a  Bunsen  flame  almost  to  the  melting 
point,  then  removed  from  the  flame  and  immediately  drawn  out  with  a  sud- 
den jerk.  Very  fine  capillary  pipettes  can  be  thus  made. 

The  removal  of  the  exudate  is  accomplished  as  follows:  a  small  cut  is 
made  with  scissors  through  the  skin  of  the  guinea-pig's  abdomen;  the  capil- 
lary pipette,  the  large  end  of  which  is  kept  closed  with  the  index  finger,  is 
forced  into  the  abdominal  cavity  with  a  single  push.  The  pressure  of  the 
finger  is  next  relaxed  and  the  tube  slowly  withdrawn.  In  order  to  avoid 
injuring  the  intestines,  the  precautions  usually  advised  in  intraperitoneal 
inoculations  should  be  observed  here.  The  author  has  found  Friedberger's 
method  of  holding  the  animal  very  serviceable  (see  Fig.  5).  The  procedure 
is  absolutely  painless,  moreover,  the  ordinarily  sensitive  guinea-pigs  with- 
stand the  operation  almost  without  uttering  a  sound. 

It  is  best  to  withdraw  the  exudate  immediately  after  the  injection  and 
then  at  intervals  of  five  to  ten,  twenty,  and  thirty  minutes,  etc.  Observations 
are  made  directly  in  hanging-drop  preparations.  Stained  specimens  are 
less  reliable  and  instructive  because,  according  to  the  investigations  of 
Radziewsky,  the  findings  are  dependent  upon  the  kind  of  coloring  matter 
used.  Bacteria  which  are  in  the  process  of  dissolution  soon  lose  the  power 
of  being  stained  by  methylene  blue,  while  they  retain  their  affinity  for 
carbol-fuchsin  and  aqueous  solution  of  gentian  violet.  The  production  of 
granules  occurs  only  incompletely  in  stained  preparations. 

The  prognosis  for  the  animal  quoad  vitam,  is  unfavorable,  if  bacterioly- 
sis does  not  occur;  good,  if  it  does.  Yet  there  are  exceptions  to  the  latter 
rule,  a  subject  to  which  reference  will  be  made  later  on.  Now  that  the 
most  important  technical  details  of  the  Pfeiffer  phenomenon  have  been  con- 
sidered, the  protocol  following  will  more  clearly  illustrate  their  procedure. 

Tit-ration  of  a  bacteriolytic  serum  (after  Pfeiffer) . 


124 


BACTERIOLYSINS  AND  HEMOLYSINS. 


Guinea-pig     1-4-07 

One  loopful  of 

+  0.1  typhoid 

Beginning  of  bacterio-       Animal 

No.  i. 

typhoid  culture. 

serum. 

lysis  after  10  minutes;     remained 

after  30  minutes  exu- 

alive. 

in  i  c.c.  of  bouillor 

i  intraperitoneally.      date  was  sterile. 

Guinea-pig     1-4-07 

One  loopful  of 

+  0.01  typhoid 

Beginning  of  bacterio- 

Animal 

No.  2. 

typhoid  culture. 

serum. 

lysis  after  15  minutes; 

remained 

, 

after  20  minutes  only        alive. 

in  i  c.c.  of  bouillon  intraperitoneally. 

isolated,      non-motile 

bacteria,  many  gran- 

ules; after  40  minutes 

. 

sterile. 

Guinea-pig    1-4-07 

One  loopful  of 

+  0.001  typhoid     Beginning  of  bacterio- 

2/4  animal 

No.  3. 

typhoid  culture. 

serum.               lysis  after  1  5  minutes  ; 

slightly  ill. 

after  20  minutes  nu- 

3/4 animal 

V 

in  i  c.c.  of  bouillon  intraperitoneally. 

merous  granules  and 

recove  red 

also  many  non-motile 

and      re- 

bacteria;   after    an 

mained 

hour,  no  bacteria  at    alive. 

all. 

Guinea-pig    1-4-07 

One  loopful  of 

+  0.0001  typhoid 

After  20  minutes  gran-    2/4  animal 

No.  4. 

typhoid  culture. 

serum. 

ules  and  many  motile 

found 

t)3.ctcrici  *  ciftcr  i  hour 

dead. 

in  i  c.c.  of  bouillon  intraperitoneally. 

motile   bacteria   very 

numerous. 

Guinea-pig     1-4-07 

One-nfth  loopful 

After  20  minutes  gran- 

2/4anima 

No.  5.                              in  i  c.c.  of 

ules  and  many  motile 

found 

bouillon 

bacteria  ;     after      i 

dead. 

intraperitoneally. 

hour  motile  bacteria 

very  numerous. 

Guinea-pig    1-4-07 

One  loopful. 

-f  o  .  i  normal 

. 

After  15  minutes  many     2/4  animal 

No.  6. 

serum. 

granules,  also  isolated  |        found 

f"1            A                      f*1      ;           A       A 

in  i  c.c.  of  bouillon. 

motile  £LnQ  non~rnotiic 
bacteria  ;    after     20 

UWAAJ  • 

minutes   motile   bac- 

teria; after  30  minu- 

tes    motile     bacteria 

very  numeours. 

Guinea-pig    1-4-07 

One  loopful  of 

+  o  .  i  typhoid 

Completely    similar 

2/4  animal 

No.  7. 

bacteria,  paraty- 

serum. 

findings. 

found 

phosus.       (Viru- 

dead. 

lence  i/io  loop- 

ful.) 

in  i  c.c.  of  bouillon. 

The  bacteriolytic  titer  of  the  tested  serum  in  this  case  would  lie  between  i  mg.  and  i/io 
mg.  and  could  be  exactly  determined  by  further  tests  which  would  take  into  consideration 
the  intermediate  doses. 


ENDOTOXIN. 


125 


On  close  study  of  the  above  experiment,  it  will  be  noted  that  even  in  those 
cases  in  which  the  animals  died  of  the  infection,  bacteriolytic  phenomena 
were  not  altogether  absent.  They  occurred  particularly  in  the  beginning 
and  were  incomplete.  This  can  be  considered  as  evidence  of  the  fact  that 
even  normal  animals  possess  a  certain  supply  of  bacteriolysins  which  are, 
however,  readily  exhausted.  This  amount  of  normal  bacteriolysin  in  serum 
varies  greatly  with  the  species  of  animal;  thus  the  sera  of  man  and  rabbit 
contain  very  little  normal  bacteriolysins  for  cholera  and  typhoid,  while 
horse's  serum  is  well  supplied  with  the  same. 

According  to  Kolle,  a  loopful  of  virulent  cholera  vibrios  is  destroyed  in  the  peritoneal 
cavity  of  a  guinea-pig,  by 

0.005  to  o.oi  c.c.  of  normal  horse's  serum, 
o.oi    to  0.02  c.c.  of  normal  ass  serum, 
o  02    to  0.03  c.c.  of  normal  goat's  serum, 
o.i      to  0.3    c.c.  of  normal  rabbit's  serum. 

The  protective  action  of  bacteriolytic  sera  differs  very  essentially  from 

that  of  antitoxic  sera.     For  the  latter,  the  law  of  multiple  proportions  holds 

true;  a  stronger  dose  of  toxin  is  neutralized  by  a  proportionately  larger 

amount  of  antitoxin;  to  bacteriolytic  sera  this  rule  does  not 

Endotoxin.    apply.     If  the  bacteria  are  increased  beyond  a  certain  quantity, 

their  dissolution  can  indeed  be  accomplished  by  the  addition  of 

sufficient  amounts  of  (bacteriolysin),  but  the  animal  dies  nevertheless.     Its 

peritoneal  cavity  examined  during  life  or  post-mortem  may  be  absolutely 

sterile.     Pfeiffer's  explanation  for  this  phenomenon  is  that  the  endotoxins 

within  the  bacteria  are  liberated  by  bacteriolysis  and  kill  the  animal.     Fatal 

results  from  endotoxin  follow  in  a  similar  manner  when  dead  instead  of 

living  bacteria  are  injected. 

Since  endotoxins  can  continue  their  effective  action  in  spite  of  the  serum, 
it  is  evident  that  the  usual  bacteriolytic  serum  lacks  the  power  to  neutralize 
the  poisons  of  the  endotoxins.  Many  investigators  have  attempted  to  sup- 
ply this  deficiency.  (This  will  be  considered  later). 

While  bacteriolysis  may  take  place  without  any  resulting  protective  action, 
on  the  other  hand  a  serum  may  be  curative  in  spite  of  the  absence  of  bacterio- 
lysis. This  is  well  demonstrated  in  Metschnikoff's  experiment. 

A  marked  leucocytosis  in  the  abdominal  cavity  of  a  guinea-pig 
Metschnikoff's  is  produced  by  the  intraperitoneal  injection  twelve  hours 
Experiment,  previously  of  5  to  10  c.c.  of  aleuronat  solution  or  sterile 
bouillon.  Pfeiffer's  experiment  is  then  performed.  As  a  rule, 
bacteriolysis  occurs  also  here  up  to  a  certain  point,  particularly  when  chol- 
era vibrios  are  used;  most  of  the  bacteria,  however,  retain  their  form  and 
are  taken  up  by  the  leucocytes. 

Metschnikoff  used  this  experiment  to  uphold  his  theory  of  the  signifi- 


126  BACTERIOLYSINS  AND   HEMOLYSINS. 

cance  of  phagocytosis.  Pfeiffer  maintained  that  bacteriolysis  was  the  most 
important  protective  weapon  of  the  immune  organism  against  bacterial  in- 
vasion. According  to  Metschnikoff  and  his  followers  among  whom  Bail 
in  particular  must  be  mentioned,  bacteriolysis  in  the  abdominal  cavity  is  only 
an  exceptional  phenomenon  (test-tube  experiment  in  vivo) ;  its  occurence  is 
made  possible  by  the  circumstance  that  the  abdominal  cavity  is  as  a  rule 
almost  free  of  wandering  cells,  and  that  the  few  which  are  present  are  so 
injured  by  the  severity  of  the  infection,  that  they  disintegrate.  If  their 
number  increases,  bacteriolysis  does  not  occur,  or  at  least  is  only  slight. 
Likewise,  bacteriolysis  is  incomplete  in  the  presence  of  cells,  for  instance  in  the 
blood,  spleen,  liver  and  subcutaneous  tissue,  etc. 

A  detailed  consideration  of  this  much  mooted  problem  does  not  fall 
within  the  compass  of  this  book.  It  is  sufficient  to  have  pointed  out  the 
great  questions  of  fundamental  significance  which  hinge  upon  the  discussion 
of  the  Pfeiffer  experiment,  questions  which  concern  the  essential  features  of 
antibacterial  immunity.  It  can  be  readily  understood,  therefore,  why  the 
phenomenon  of  bacteriolysis  has  been  so  much  studied,  although  its  practical 
significance  is  only  limited. 

The  Pfeiffer  experiment  can  be  used  in  the  differentiation  of 
The  Practical  bacteria  as  well  as  in  the  demonstration  of  bacteriolysins  in 
Application  of  serum.  It  serves  as  a  control  for  the  agglutination  reaction. 

Pfeiffer  and  Kolle,   Briefer  and  others,   have  used  bacteriolysis   as  a 
Experiment. 

method  of  estimating  the  immunity  obtained  by  active  protective  im- 

*  munization  against  cholera  and  typhoid  in  man.  It  must,  however,  be 
questioned  whether  it  is  admissible  to  draw  conclusions  as  to  the  degree  of  active  im- 
munity from  the  height  of  the  bacteriolytic  titer  of  the  serum,  inasmuch  as  animals 
are  found  which  possess  no  active  immunity  and  still  have  sera  of  high  bacteriolytic 
powers. 

The  most  important  practical  use  of  the  Pfeiffer  experiment  lies  in  the  identi- 
fication of  suspected  cholera  cultures.  In  Germany,  the  Pfeiffer  test  made 
with  the  vibrios  obtained  in  pure  culture  from  the  suspected  patients,  is 
required  for  the  official  diagnosis  of  the  first  cases  of  cholera. 

The  serum  used  for  this  purpose  should  be  at  least  strong  enough  in 
amounts  of  0.0002  c.c.  to  cause  the  disintegration  of  the  bacteria  in  one 
hour,  when  a  mixture  of  one  loopful  of  an  eighteen-hour  agar  culture  of 
cholera  with  i  c.c.  of  nutrient  bouillon  is  injected  into  the  peritoneal  cavity 
of  a  guinea-pig. 

For  this  experiment  four  guinea  pigs  of  250  grams  weight  are  used. 


IDENTIFICATION    OF    CHOLERA   CULTURES. 


127 


Animal.                 Culture. 

Serum                     Method  of              Result  in  cholera 

injection.                         cases. 

No    i 

One  loopful  of  an 

I 
o.ooi    c.c.    cholera    Intraperitoneally.    After  20  minutes  or  at 

1  8  hours'growth  of 

serum  =  5  times 

the     latest     i    hour 

culture    suspected 

the  titer  dose. 

bacteriolysis    occurs  ; 

to  be  cholera  in  i 

animal  remains  alive. 

c.c.  of  bouillon. 

No.  2  

One  loopful  of  an  1  8 

0.002    c.c.    cholera 

Intraperitoneally.    After  20  minutes  or  at 

hours'    growth   of 

serum  =  10    times 

the    latest    i    hour, 

culture    suspected 

the  titer  dose. 

bacteriolysis  occurs; 

to  be  cholera  in  i 

animal  remains  alive. 

c.c.  of  bouillon. 

t  I 

p* 

No.  3  (con- 

One loopful  of  an  1  8 

o.oi  c.c.  of  normal 

Intraperitoneally.    Increase  in  number  of 

trol.)               hours'    growth   of 

serum  =  50     times 

1  "  bacteria  ;  animal  dies. 

culture    suspected 

the    titer   dose   of 

. 

to    be    cholera   in 

the  immune  serum. 

i  c.c.  of  bouillon. 

No.  4  (con- 

One-fourth loopful 

Intraperitoneally.    Increase  in  number  of 

trol  of  vir- 

of   an  ,  1  8    hours' 

'    bacteria,  animal  dies. 

ulence   of 

growth    suspected 

culture.) 

of  being  cholera  in 

\ 

i  c.c.  of  bouillon. 

In  cases  of  subsiding  cholera,  the  Pfeiffer  experiment  is  performed  with 
the  serum  of  the  patient  in  dilutions  of  i  to  20,  i  to  100  and  i  to  500. 

Bacteriolysis  with  typhoid  organisms  is  less  typical  than  with  cholera. 
For  diagnostic  purposes  the  test  is  resorted  to,  only  when  the  agglutination 
reactions  are  doubtful.  When  bacteriolysis  also  gives  uncertain  results,  an 
animal  is  immunized  with  the  typhoid  suspected  bacteria  and  its  serum 
tested  for  its  power  of  agglutinating  or  destroying  definitely  known  typhoid 
bacteria  and  eventually  the  immunized  animal  may  be  injected  with  virulent 
typhoid  bacilli. 

Bacteriolysis  is  even  more  unsatisfactory  with  bacillus  paratyphosus  and 
the  related  hog  cholera  group  of  organisms. 

While  in  typhoid  the  onset  of  bacteriolysis  offers  a  favorable  prognosis  for  the  animal, 
guinea-pigs  inoculated  with  bacteria  of  the  paratyphoid  hog-cholera  group  die  in  spite 
of  complete  bacteriolysis.  Death  always  takes  place  late  (from  three  to  six  days), 
while  the  control  animals  succumb  in  about  twenty-four  hours.  Bacteriolysis  has  also 
been  observed  with  the  bacillus  of  dysentery  and  with  the  tubercle  bacillus;  but  thus 
far,  these  phenomena  have  gained  no  clinical  significance.  Bacteriolysis  does  not  occur  in 
anthrax,  pest  and  the  various  diseases  due  to  cocci. 


128  BACTERIOLYSINS  AND  HEMOLYSINS. 

II.  Bactericidal  Plate-culture-method. 

(Plattenverfahren)  after  Neisser  and  Wechsberg. 

For  the  determination  of  the  bactericidal  titer  of  a  serum,  Neisser  and 
Wechsberg  recommended  the  so-called  bactericidal  plate-culture  method.  The 
principle  of  it  is  as  follows:  the  serum  to  be  tested  is  inactivated;  different 
amounts  of  this  inactivated  serum  are  mixed  with  a  definite  constant  quan- 
tity of  bacteria,  and  a  constant  quantity  of  active  normal  serum  is  added  as 
complement.  This  mixture  is  left  in  the  thermostat  sufficiently  long  to  per- 
mit the  occurrence  of  bacteriolysis.  Now,  to  determine  whether  and  to 
what  degree  death  of  bacteria  resulted  from  the  effect  of  the  reactivated 
bacteriolysins  (or  of  some  bactericidal  substance  otherwise  unknown),  agar 
is  added,  the  mixture  plated,  and  the  number  of  colonies  counted. 

Stern  and  Korte  recommend  this  procedure  for  clinical  purposes,  as  a 
substitute  for  the  Pfeiffer  test  in  the  diagnosis  of  typhoid.  They  point  out 
the  sparing  of  animals  as  one  of  its  advantages.  On  the  other  hand,  this 
method  consumes  much  more  time  and  its  results  are  less  trustworthy.  It 
has  not  found  a  place,  therefore,  in  clinical  practice. 

The  technique  of  Stern  and  Korte  is  the  following:  the  serum 
The  Technique  °f  tne  patient,  and  that  of  a  person  not  ill  with  typhoid  as 

of  the        control,  are  inactivated  for  one-half  hour  at  56°  C.  and  i  c.c. 

Method.  of  each  in  decreasing  dilutions  is  poured  into  sterile  test-tubes. 
To  each  is  added  0.5  c.c.  of  a  twenty-four  hour  typhoid  bouil- 
lon culture  diluted  in  bouillon  to  i  :  5000  or  i  :  10000.  For  reactivation  0.5 
c.c.  of  fresh  normal  rabbit's  serum  in  a  dilution  of  i  to  12  in  physiological 
saline  is  added  and  the  whole  thoroughly  shaken.  The  tubes  are  then 
placed  into  the  thermostat  for  three  hours.  The  entire  contents  of  each 
mixture  is  plated  in  agar,  and  after  eighteen  to  twenty-four  hours  the  plates 
are  to  be  examined.  That  particular  plate  is  considered  to  indicate  the  end 
value  of  the  bacteriolytic  action  of  the  serum  in  which  there  is  evident  a  very 
great  decrease  in  the  number  of  colonies  as  compared  with  the  innumerable 
colonies  found  on  the  control  plates. 

Certain  other  controls  are  necessary: 

1.  One  tube  containing  culture  and  complement. 

2.  One  containing  culture  and  inactivated  immune  serum  in  the  highest 
concentration  used. 

3.  The  same  with  inactivated  normal  serum  instead  of  immune  serum. 

4.  Complement  without  culture  and  immune  serum  to  test  its  sterility. 

5.  Immune  serum  without  culture  and  complement  to  test  its  sterility. 

6.  One  tube  containing  only  culture,  to  be  plated  immediately. 

7.  One  tube,  containing  only  the  culture,  to  be  plated  after  standing  in 
the  thermostat  for  three  hours. 


BACTERICIDAL    PLATE-CULTURE-METHOD. 


I2Q 


Topfer  and  Jaffe  pour  a  thin  layer  of  agar  into  a  petri  dish  and  let  it 
harden.  Upon  this  the  culture-serum-agar  mixture  is  poured,  and  after 
hardening  is  covered  with  another  thin  layer  of  agar.  In  this  way  the 
formation  of  a  film  of  culture  in  the  water  of  condensation  is  avoided. 

A  practical  example  is  appended  to  illustrate  the  plate  culture  method. 


Result  (poured  after  remaining  3 

hours  in  the  thermostat). 

Culture. 

Serum. 

Complement. 

Normal  serum. 

Immune  serum. 

0.5  1/5000  typh. 

I/IOO           C.C. 

0.5     :i2  rabbit's. 

o  colonies. 

Many  thousand. 

0.5  1/5000  typh. 

1/500        c.c. 

0.5    :i2  rabbit's. 

100  colonies. 

Many  thousand. 

0.5  1/5000  typh. 

I/IOOO        C.C. 

0.5     :i2  rabbit's. 

Many  thousand. 

Many  thousand. 

0.5  1/5000  typh. 

1/5000      c.c. 

0.5    :i2  rabbit's. 

oc 

Many  thousand. 

0.5  1/5000  typh. 

I/IOOOO     C.C. 

0.5    :i2  rabbit's. 

oc 

500 

0.5  1/5000  typh. 

I/2OOOO      C.C. 

0.5  1:12  rabbit's. 

oc 

200 

0.5  1/5000  typh. 

1/30000    c.c. 

0.5  1:12  rabbit's. 

Q 

0.5  1/5000  typh. 

1/40000    c.c. 

0.5  1:12  rabbit's. 

0.5  1/5000  typh. 

1/50000    c.c. 

0.5  1:12  rabbit's. 

60 

0.5  1/5000  typh. 

I/IOOOOO  C.C. 

0.5  1:12  rabbit's. 

800 

0.5  1/5000  typh. 

I/2OOOOO  C.C. 

0.5  1:12  rabbit's. 

OC 

Control  I 

— 

0.5  1:12  rabbit's. 

Many  thousand. 

0.5  1/5000  typh. 

Control  II  and  III 

I/IOO           C.C. 

— 

oc 

oc 

0.5  1/5000  typh. 

Control  IV- 

—  . 

0.5  1:12  rabbit's. 

0 

Control  V  - 

I/IOO           C.C. 

— 

0 

Control  VI. 

o.  5  1/5000  typh. 

— 

— 

Many  thousand. 

immediately  poured. 

Control  VII 

— 

— 

oc 

0.5  1/5000  typh. 

poured  after  3  hours. 

In  addition  to  the  results  which  one  would  expect,  this  experiment  shows  one  striking 
point.  With  the  normal  serum  the  tube  which  contains  the  largest  amount  of  normal 
bacteriolysins  shows  on  plating,  the  fewest  germs.  The  greater  the  dilution  of  the  serum 
the  more  prolific  is  the  bacterial  growth.  The  titer  of  the  normal  serum  in  this  case  lies 
between  i/ioo  and  1/500.  The  controls  show  that  the  serum  and  complement  are 
sterile,  and  that  the  inactive  normal  serum  is  ineffective.  During  the  three  hours  in  the 
thermostat  the  bacterial  suspension  has  become  stronger.  The  retarded  growth  in 
the  complement  culture  tube  can  be  traced  probably  to  the  presence  of  normal 
bacteriolysins. 

With  the  immune  serum  on  the  other  hand,  results  are  quite  different.     Where 
the  most  concentrated  serum  is  used,  the  bacterial  growth  is  still  rather  profuse;  only 
the  moderate  doses  show  a  true  bactericidal  action  and  the  small  doses  are  altogether 
ineffective.     The  titer  of  this  serum  is  between  1/3000  and  1/4000. 
9 


130 


BACTERIOLYSINS  AND  HEMOLYSINS. 


Neisser  and  Wechsberg  explain  this  phenomenon  by  the  so-called 
"deviation  of  the  complement"  They  assume  that  in  the  serum  of  higher 
concentration  there  are  so  many  amboceptors  that  the  bacteria  cannot  bind 
them  all.  The  amboceptors  remaining  free  attach  themselves  to  the  com- 
plement by  means  of  their  complementophile  group  just  as  the  already 
bound  amboceptors  have  done.  Thus,  a  part  of  the  complement  is  deviated 
from  the  bacteria  and  only  an  incomplete  bacteriolysis  takes  place. 

The  theory  of  complement  deviation  does  not  in  the  opinion  of  the  author  withstand 
critical  examination.  Particularly  the  evidence  brought  forward  by  Bordet  and  Gengou 
that  the  affinity  of  complement  for  the  bacterium  +  amboceptor  complex  (Sensitized 
bacterium)  is  considerably  greater  than  for  free  amboceptor,  militates  against  the  view 
of  Neisser  and  Wechsberg. 

It  is  possible  that  agglutination  may  account  for  the  phenomenon  of  deviation  of  the 
complement  in  that  the  agglutinated  masses  of  bacteria  afford  a  more  resistant  barrier 
to  the  action  of  the  bacteriolysins.  The  author  has  now  and  then  observed  an  analogous 
phenomenon  in  hemolytic  experiments;  strong  doses  of  hemolysin  were  less  effective 
than  moderate  ones,  and  in  these  cases  the  momentary  hemagglutination  was  readily 
visible.  Also,  by  titrating  bactericidal  sera  in  animal  experiments,  it  has  been  found 
that  moderate  doses  often  afforded  the  greatest  protective  action. 

For  the  practical  application  of  the  plate  culture  method,  knowledge  of 
the  following  data  is  important,  as  it  is  necessary  to  consider  the  difference 
between  the  bactericidal  titer  of  sera  of  normal  and  of  typhoid  patients. 
According  to  Korte  and  Steinberg  the  bactericidal  titer  was 


Of  normal  cases 

Of  typhoid  cases 

Under  i  oo  in                                     

74      per  cent 

o  o  per  cent 

Between  100  and  1000  in         

8  6  per  cent 

•3  o  per  cent. 

Between  1000  and  10  ooo  in            .  .  .    

154  per  cent 

15  i  per  cent. 

Between  10  ooo  and  100  ooo  in       

2  o  per  cent 

23  3  P^r  cent. 

Over  100  ooo  in     

o  o  per  cent 

58  3  per  cent. 

The  bactericidal  titer  does  not  run  strictly  parallel  either  with  aggluti- 
nation or  the  Pfeiffer  experiments.  It  falls  toward  the  end  of  the  disease 
and  is  low  during  convalescence. 

Besides  being  used  in  typhoid,  the  plate  culture  method  has  been  employed  for  experi- 
mental purposes  in  cholera  and  dysentery;  in  these  diseases,  however,  it  possesses  no 
clinical  diagnostic  significance. 

Concerning  bacillus  paratyphosus,  the  views  of  different  authorities  are  widely  at 
variance.  While  some  obtained  very  good  results,  similar  to  those  found  in  typhoid 
fever,  Topfer  and  Jaffe  could  demonstrate  no  bactericidal  power  whatever  in  vitro. 
This  difference  can  be  explained  only  by  the  differences  in  sera. 


HEMOLYSINS.  131 

Hemolysins. 

An  animal  that  is  injected  with  the  red  blood  cells  of  a  different  species, 
develops  in  its  serum  antibodies  which  are  biologically  analogous  to  bac- 
teriolysins  and  differ  from  them  only  in  that  they  cause  disintegration  of 
erythrocytes  instead  of  bacteria.  These  antibodies  are  therefore  called 
hemolysins,  or  to  be  more  precise  immune-hemolysins,  since  they  arise 
through  a  process  of  immunization.  The  breaking  up  of  the  red  blood 
corpuscle,  hemolysis,  is  recognized  by  the  naked  eye.  The  hemoglobin 
passes  from  the  erythrocytes  into  the  surrounding  fluid  (serum  or  physiolog- 
ical salt  solution)  and  colors  it  red.  The  previously  opaque  blood,  lakes 
and  becomes  transparent.  Immune-hemolysins  like  bacteriolysins  belong 
to  the  class  of  amboceptors.  They  are  relatively  thermolabile  in  that  they 
withstand  a  temperature  of  from  56°  to  58°  C.  without  being  injured,  and 
they  require  complement  for  the  development  of  their  hemolytic  action. 
Furthermore,  immune-hemolysins  like  all  amboceptors,  are  specific,  i.e., 
the  serum  of  a  rabbit  immunized  against  horse's  blood  can  dissolve  only  the 
blood  of  a  horse  and  not  that  of  a  hen  or  cow.  On  the  other  hand,  group 
reactions  occur  here  also;  for  instance  the  immune-hemolysin  produced  in  a 
rabbit  against  horse's  blood  is  likewise  active  against  donkey's  blood. 

Just  as  various  antitoxins,  agglutinins,  precipitins  and  bac- 

Normal       teriolysins  can  be  found  in  normal   serum,  so  also  normal 

Hemolysin.    hemolysins  of  amboceptor  structure  can  be  discovered  in  the 

blood  of  many  species  of  animals  or  individual  animals. 
While  normal  hemolysins  come  into  play  in  only  a  few  reactions,  as  in 
several  modifications  of  the  Wassermann  test,  the  significance  of  immune- 
hemolysins  is  extraordinarily  great.  These  antibodies,  discovered  by 
Bordet,  and  independently  by  von  Dungern  and  Landsteiner,  were  carefully 
studied  by  Ehrlich  and  Morgenroth  and  many  others.  Such  researches 
have,  first  of  all,  greatly  advanced  the  subject  of  immunity  in  its  theoretical 
aspects,  in  that  they  have  created  the  possibility  for  the  discovery  in  minute 
detail  the  finer  relationship  which  has  explained  some  of  the  phenomena 
occurring  in  bacteriolysis.  Furthermore,  the  studies  of  hemolysins  led  to  the 
discovery  of  the  complement  fixation  method,  a  procedure  of  exceptional 
practical  value. 

As  far  as  the  technique  for  obtaining  immune-hemolysins  is 

Production     concerned,  the  rules  which  hold  for  every  process  of  immu- 

of  Hemolytic  nization  are  naturally  to  be  followed  here  also.     It  is  not 

Sera.         possible,  however,  to  immunize  every  kind  of  animal  against 

every  type  of  red  blood  corpuscles.  Rabbits,  goats,  horses 
and  chickens  are  the  ones  which  are  best  adapted  to  supply  hemolytic  sera. 
An  animal  produces  a  better  hemolysin  the  remoter  its  relationship  to  the 
animal  from  which  the  erythrocytes  for  injection  are  taken.  The  blood  to 


132  BACTERIOLYSINS  AND  HEMOLYSINS. 

be  injected,  can  be  employed  in  just  the  condition  in  which  it  flows  from  the 
vein.  Nevertheless  it  is  as  a  rule  defibrinated,  to  prevent  coagulation. 
The  simplest  and  most  practical  way  of  doing  this  is  to  place  some  glass 
beads  into  a  bottle  or  Erlenmeyer  flask  and  then  sterilize  by  dry  heat.  The 
blood  coming  from  the  vein  is  allowed  to  flow  into  one  of  these  flasks  and 
then  it  is  repeatedly  shaken  for  several  minutes.  This  suffices  to  defibrinate 
the  blood  and  thus  prevent  coagulation. 

The  production  of  hemolysins  depends  entirely  upon  the  red  blood 
corpuscles.  The  presence  of  the  serum  is  not  only  superfluous,  but  even 
harmful,  as  experience  has  shown  that  dangerous  reactions  may  follow 
the  injection  of  foreign  serum. 

Before    injecting,    therefore,    the    erythrocytes    are    washed.     For    this 
Washing  of    purpose  a  few  cubic  centimeters  of  defibrinated  blood  are  poured  into 
Red  Blood     a  centrifuge  tube  and  the  level  of  the  fluid  marked  on  the  tube.     An 
Corpuscles,     equal  or  double  this  amount  of  0.85  per  cent,  saline  is  added,  and  the 
tube  rapidly  centrifugalized.     The  erythrocytes  fall  to  the  bottom,  while 
the  upper  layers  of  the  tube  consist  of  diluted  serum  more  or  less  tinged  with  hemoglobin. 
The  fluid  is  carefully  decanted,  fresh  saline  added,  the  tube  shaken,  and  again  centrifu- 
galized.    If  this  is  done  two  to  three  times  the  erythrocytes  can  be  freed  of  the  last 
traces  of  serum;  finally,  by  adding  saline  up  to  the  mark  made  at  the  beginning  of  the 
experiment  the  erythrocytes  are  obtained  in  the  normal  concentration,  just  as  in  the 
blood,  but  completely  free  of  serum. 

The  injection  of  the  washed,  defibrinated  blood,  can  be  af- 
Immunization.  fected  subcutaneously,  intravenously,  or  intraperitoneally. 

With  the  subcutaneous  and  intraperitoneal  methods  in  a  rab- 
bit, injections  of  from  5  to  20  c.c.  are  necessary  at  intervals  of  five  to  six  days. 
Far  larger  quantities  should  be  given  to  bigger  animals,  like  goats  and 
sheep.  Subcutaneous  injections  often  cause  infiltrations  and  occasionally 
abscesses.  The  author  therefore  uses  the  intravenous  method  exclusively 
in  rabbits. 

A  suspension  of  washed  blood  corpuscles  is  diluted  four  to  five  times  with  physiological 
saline;  0.5  to  i.o  c.c.  of  this  fluid  is  slowly  injected  into  the  ear  vein  every  five  to  six 
days.  Three  injections  are  almost  always  sufficient  for  procuring  a  good  serum.  The 
animals  sustain  the  first  two  injections  with  ease,  but  the  third  and  following  ones  are 
not  altogether  without  danger.  This  is  supposed  to  be  akin  to  anaphylactic  phenomena. 
It  is  therefore  advisable  to  immunize  several  animals  simultaneously,  so  that  in  case  one 
dies  there  is  another  to  replace  it.  Furthermore  there  are  such  marked  individual  varia- 
tions in  the  ability  to  produce  hemolysins  that  it  is  best  to  have  several  animals  to  choose 
from.  Beginning  on  the  sixth  day  after  the  third  injection,  blood  should  be  withdrawn 
for  the  determination  of  the  hemolytic  strength  and  this  process  repeated  daily  until 
the  titer  has  reached  a  satisfactory  height  and  then  the  animal  should  be  bled.  If  only 
a  small  amount  of  hemolysin  is  needed,  the  animal  can  be  allowed  to  live;  it  will  gradually 
lose  its  titer  completely  and  will  act  apparently  like  a  normal  animal.  Nevertheless, 
an  essential  difference  exists.  For  if  the  animal  previously  immunized  is  again  injected, 
hemolysins  reappear  after  a  short  incubation  period,  whereas  in  a  normal  animal  a 
prolonged  immunization  is  necessary.  Hemolysins,  therefore,  exist  to  a  certain  extent 


PRESERVATION    OF  HEMOLYSINS.  133 

in  a  preformed  state  in  the  cells  of  an  immunized  animal.  If  a  stimulus  to  immunization 
occurs,  the  hemolytic  substances  are  thrown  off  into  the  circulation,  while  in  a  normal 
animal  the  formation  of  hemolysins  by  the  cells  must  first  take  place. 

If  a  great  amount  of  hemolysin  of  the  same  titer  is  needed,  it  is 
ThePreser-  best  to  bleed  the  animal  to  death.  For  the  preservation  of 

vation  of      hemolysins  the  author  recommends  the  following  procedure 

Hemolysins.    which  he  has  found  very  trustworthy.     One  to  3  c.c.  of  serum 

obtained   sterile,   are   poured   into   sterile   tubes,   which   are 

closed  with  absorbent  cotton.     The  tubes  are  placed  into  a  water  bath  at 

56°  C.  for  one-half  hour  to  inactivate  the  serum  and  are  then  covered  with 

sterile  rubber  caps.     (These  are  sterilized  by  placing  them  in  a  i  per  cent. 

sublimate  solution  for  forty-eight  hours). 

An  immune  hemolysin  must  answer  both  qualitative  and  quantitative 
determinations;  qualitative,  whereby  is  proven  that  the  serum  can  dissolve 
only  the  red  blood  cells  which  serve  as  antigen  or  to  a  slight  degree  those  of 
nearly  related  animals,  and  that  it  has  only  the  effect  of  a  normal  serum  upon 
the  erythrocytes  of  other  animals.  The  quantitative  estimation  supplies 
the  only  means  for  the  absolute  differentiation  between  a  normal  and  an 
immune  serum.  In  complement  fixation  where  hemolysis  bears  an  active 
part,  it  is  the  quantitative  use  of  the  hemolysin  which  decides  the  result  of  the 
reaction.  The  immune  serum  must  therefore  be  "titrated." 

If  fresh  active  hemolytic  immune  serum  is  used,  a  constant  quantity  of 
blood  serving  as  antigen  is  mixed  with  decreasing  quantities  of  serum  and 
the  mixtures  placed  into  the  thermostat.  Results  like  the  following  will  be 
obtained. 


Antigen  blood. 


Hemolytic  serum  of  immune  rabbit,    j     Result  after  2  hours. 


i  c.c.  of  5%  sheep's  blood |  i  c.c.  of  active  serum,  i  to  10 Hemolysis. 

i  c.c.  of  5%  sheep's  blood j  i  c.c.  of  active  serum,  i  to  20 Incomplete  hemolysis. 

i  c.c.  of  5%  sheep's  blood i  i  c.c.  of  active  serum,  i  to  50 Incomplete  hemolysis. 

i  c.c.  of  5%  sheep's  blood i  c.c.  of  active  serum,  i  to  100 No  hemolysis. 


On  the  basis  of  this  experiment  the  titer  of  the  hemolytic  serum  for 
sheep's  blood  would  lie  between  i/io  and  1/20.  But  this  is  incorrect,  as  it 
was  pointed  out  previously  that  by  immunization  only  the  amboceptors 
are  increased  and  the  complement  remains  unchanged.  Each  of  the  above 
dilutions  decreases  therefore  not  only  the  amount  of  hemolysin,  the  quanti- 
tative estimation  of  which  is  the  object  of  the  experiment,  but  also  the 
complement.  Inasmuch  as  the  latter  was  not  at  first  increased,  a  point  is 
soon  reached  where  there  is  no  complement  at  all  in  the  diluted  fluid;  as  a 
result  hemolysis  cannot  occur,  for  only  the  combination  of  hemolysin  + 
sufficient  complement,  can  exhibit  any  hemolytic  action.  Correct  titration 


134  BACTERIOLYSINS  AND   HEMOLYSINS. 

consists  therefore  in  allowing  varying  quantities  of  hemolysin  with  a  constant 
amount  of  complement  to  act  upon  a  constant  quantity  of  red  blood  cells.  The 
simplest  method  of  accomplishing  this  is  first  to  destroy  the  complement  by  in- 
activation  of  the  hemolytic  serum,  then  to  make  the  desired  dilutions,  and  finally 
to  add  to  all,  the  same  amount  of  normal  serum  as  complement.  The  normal 
serum  of  an  animal  of  the  same  species  as  that  which  provided  the  immune 
serum  can  under  no  circumstances  serve  as  complement.  On  the  contrary, 
foreign  sera  are  much  more  suitable;  and  guinea-pig's  serum  is  especially 
recommended  as  complement,  when  immune  rabbit's  serum  is  used.  Not 
every  complement  serves  equally  well  for  any  immune  serum. 

A  very  good  hemolytic  system  which  is  almost  exclusively  used  for  the 
complement  fixation  reaction,  is  sheep's  blood  as  antigen,  rabbit's  immune 
hemolysin  as  amboceptor  and  normal  guinea-pig's  serum  as  complement. 
The  preparation  of  these  ingredients  should  be  carried  out  as  follows : 

i.  Sheep's  Blood. — This  should  be  defibrinated  and  washed.  Washing 
is  necessary  because  fresh  sheep's  blood  contains  complement;  and  if  the 
blood  is  a  few  days  old,  washing  is  even  more  important. 

Although  serum  which  is  not  fresh  does  not  contain  sufficient  active 
Comple-  complement  to  cause  the  danger  of  superfluous  complement,  it  never- 
mentoids.  theless  contains  substances  which  interfere  with  hemolysis.  Probably 

the  existence  of  "complementoids"  is  the  disturbing  factor.  It  must 
be  assumed  that  complement  is  composed  of  two  biologically  different  parts,  as  is  the  case 
with  toxins  and  ferments.  One  is  the  haptophore  group,  which  has  affinity  for  the  com- 
plementophile  group  of  the  amboceptor  and  is  the  more  stable  of  the  two.  The  other 
corresponds  to  the  energy  group  of  the  toxins  (toxophore  element)  and  of  the  ferments. 
Just  as- after  the  destruction  of  the  toxophore  group  there  remain  only  innocuous  toxoids 
whose  single  perceptible  activity  consists  in  their  ability  to  neutralize  antitoxins,  so  also, 
after  the  destruction  of  the  weakly  resistant  energy  elements  of  the  complement,  there 
remain  complementoids  which  lack  the  ability  to  activate  a  bacteriolytic  or  hemolytic 
amboceptor,  although  by  virtue  of  their  uninjured  haptophore  groups  they  bind  the 
complementophile  groups  of  the  amboceptors.  In  this  way  they  usurp  the  place  of 
whatever  active  complement  may  still  be  present,  rendering  the  latter  inactive,  and  as  a 
result  hemolysis  is  absent  or  incomplete. 

Following  the  technique  of  Ehrlich  and  Morgenroth,  a  5  per  cent,  sus- 
pension of  washed  red  blood  corpuscles  is  employed  to  test  a  hemolysin.  A 
pipette,  closed  at  the  top  by  pressure  of  the  index  finger  is  thrust  to  the  bot- 
tom of  the  washed  erythrocytes  contained  in  the  centrifuge  tube;  a  definite 
amount,  for  instance  i  c.c.  is  withdrawn  and  allowed  to  flow  into  a  graduate. 
For  diluting  purposes  (in  this  case  up  to  20  c.c.)  only  isotonic  or  weakly 
hypertonic  NaCL  solutions  may  be  used.  If  water,  hypotonic  or  strongly 
hypertonic  salt  solutions  are  employed,  the  red  blood  cells  disintegrate. 
This  is  not  a  true  biological  hemolysis,  but  depends  upon  physical  basis. 
0.85  per  cent,  saline  is  most  suitable  for  the  majority  of  erythrocytes  (man, 
rabbit,  guinea-pig,  ox,  sheep).  When  instead  of  an  isotonic  salt  solution, 
an  isotonic  sugar  solution  is  made,  the  red  cells  are  retained  in  their  proper 


PRESERVATION   OF  HEMOLYSINS.  135 

form,  but  the  addition  of  hemolysin  and  complement  produces  no  hemolysis. 
The  presence  of  salt  is  indispensable  for  hemolysis  as  well  as  agglutination. 
Undiluted,  unwashed,  defibrinated  blood  if  removed  sterile  can  be  kept 
several  days  in  the  ice-box.  The  "Frigo"  apparatus  is  unsuited  for  this 
purpose,  because  the  thawing  of  the  frozen  blood  breaks  the  capsule  of  the 
red  blood  corpuscle.  The  deterioration  of  the  preserved  blood  is  recognized 
by  the  large  hemoglobin  content  of  the  serum  or  the  violet  color  of  the  blood. 

Occasionally  blood  left  in  an  ice-box  becomes  dark.  This  is  due  to  the  lack  of  oxygen. 
When  the  5  per  cent,  suspension  is  made  and  thoroughly  shaken,  the  red  color  returns. 
Such  blood  of  course  is  perfectly  serviceable. 

Still,  it  is  best  not  to  keep  blood  longer  than  four  days.  Blood  older  than  that,  even 
if  apparently  unchanged,  possesses  a  lowered  resistance  and  can  give  a  far  higher  titer 
in  hemolysin  tests  than  fresh  blood. 

2.  The  rabbit's  hemolysin  must  be  inactivated  for  one-half  hour  at  56° 
C.  if  it  is  not  kept  ready  for  use  in  an  inactivated  state.     Dilutions  are  made 
with  physiological  saline. 

3.  Guinea-pig's  complement  is  obtained  by  bleeding  to  death  a  healthy 
normal  animal. 

The  blood  is  allowed  to  flow  directly  into  a  centrifuge  tube  and  then  to  clot;  the  clear 
serum  is  obtained  by  centrifugalization.  For  hemolysin  titration  it  is  best  to  use  a 
constant  dose  of  complement  as  i  c.c.  of  a  i/io  dilution.  Complement  can  be  kept  for 
twenty-four  hours  in  the  ice-box.  When  older  than  this  it  suffers  a  distinct  decrease  in 
efficiency  as  complementoid  is  produced.  (See  above).  In  the  "Frigo,"  complement 
may  be  kept  for  weeks.  Stern,  however,  does  not  recommend  complement  preserved 
in  "Frigo"  for  use  in  complement  fixation  tests,  as  its  affinity  for  amboceptor  is  noticeably 
decreased. 

One  c.c.  of  each  of  the  three  reagents  (each  so  diluted  with  saline  that 
the  desired  dose  is  contained  within  i  c.c.)  is  mixed  and  2  c.c.  of  0.85  salt 
solution  is  added  to  make  the  total  volume  up  to  5  c.c. 

The  following  controls  are  absolutely  necessary. 

1.  A  test  tube  showing  that  hemolysin  without  complement  in  strong 
dosage  is  ineffective; 

2.  A  test  tube  indicating  that  complement  without  hemolysin  in  the 
dosage  used  is  ineffective; 

3.  A  test  tube  which  shows  that  the  NaCL  solution  is  isotonic. 

The  three  reagents  must  be  thoroughly  mixed  by  careful  shaking  of 
the  tubes  which  are  then  placed  into  the  thermostat  at  37°  C.  and  hemolysis 
watched  for.  The  duration  of  the  observation  is  a  matter  of  personal 
preference.  Only  the  length  of  time  must  always  be  mentioned.  One 
must  say,  for  instance,  that  the  titer  of  this  hemolysin  is  i  :8oo,  using  o.i  c.c. 
of  complement  under  observation  for  one-half  hour,  or  it  is  1 11500  with  o.i 
complement  under  observation  for  two  hours.  The  time  in  which  hemo- 
lysins  work  is  very  different.  While  many  hemolysins  of  the  same  titer  act 


i36 


BACTERIOLYSINS  AND  HEMOLYSINS. 


in  a  few  moments,  others  require  two  hours.  The  author  has  made  it  a 
rule  to  read  the  result  after  two  hours  observation,  but  he  notes  the  progress 
of  the  reaction  every  one-half  hour  in  order  to  determine  whether  it  is  a 
slowly  or  rapidly  acting  hemolysin. 

The  following   chart   demonstrates  the  titration  of   a  hemolysin  as  a  preliminary 
experiment  to  the  complement  fixation  method. 


Antigen. 

Amboceptor.        ,  Complement. 

! 

0.85% 
Saline 

Rest 
After  \  hr. 

lit  of  hemol 
After  i  hr. 

fsis. 
After  2  hrs. 

I. 

i  c.c.  of  5% 

i  c.c.  dilution  i  :  10 

i  c.c.  dilu- 

2 C.C. 

complete 

complete 

complete 

sheep's  blood 

tion  i  :  10 

2. 

M 

IOO 

2  C.C. 

complete 

complete 

complete 

3- 

" 

250 

2  C.C. 

complete 

complete 

complete 

4- 

U 

500 

2  C.C. 

Incom- 

Almost 

complete 

s. 

11 

750 

2  C.C. 

plete 
Almost  o 

complete 
Incom- 

complete 

6. 

it 

IOOO 

2  C.C. 

o 

plete 

complete 

Incom- 

7- 

11 

I   1500 

(i 

2  C.C. 

0 

plete 

Incom- 

0 

plete 

8. 

(1 

"                 I       2000 

" 

2  C.C. 

o 

o 

0 

Control     I 

(1 

I       10 

|,rr. 

o                     o 

o 

Control   II 

« 

i  c.c.  dilu- 

T.  C.C. 

o 

o 

o 

ion  i  :  10 

O  *" 

Control  III 

U 

4  c.c. 

o 

o 

o 

Determining  the  end  reaction  is  a  source  of  difficulty  for  the  beginner. 
Between  the  extreme  "o"  i.e.,  entire  absence  of  hemolysis,  where  the 
appearance  of  the  tube  corresponds  to  that  of  control  III  representing  a 
suspension  of  red  blood  cells  diluted  with  isotonic  saline  and  the  other 
extreme  "complete,"  i.e.,  complete  hemolysis,  where  every  trace  of  cor- 
puscular elements  has  disappeared  and  a  fluid  looking  like  dilute  red-wine 
remains,  there  are  numerous  intermediate  stages.  These  intervening 
grades  of  reaction  are  represented  by  the  terms  almost  o,  incomplete, 
almost  complete,  and  similar  expressions.  The  meaning  of  the  terms  is 
self-evident.  How  any  particular  tube  is  to  be  designated  is  of  course  a 
subjective  question  since  the  so-called  transitional  stages  are  so  large  in 
number. 

A  few  hours  after  the  reaction  is  ended,  a  remarkable  difference  may  be 
noted  between  the  tubes  in  which  hemolysis  has  occurred  and  those  in  which 
hemolysis  has  been  incomplete  or  totally  absent.  In  the  last  mentioned, 
the  red  blood  cells  had  sunk  to  the  bottom  and  above  them  remains  a  clear 
fluid  which  consists  of  pure  saline  or  diluted  serum  (complement  +  immune 
serum)  and  is  colored  accordingly.  If  the  supernatant  fluid  is  richer  in  he  mo- 
globin  than  that  of  the  corresponding  control,  it  is  evident  that  some  of  the 
erythrocytes  were  hemolysed  and  their  hemoglobin  set  free.  If  the  erythro- 
cytes  have  collected  at  the  bottom  apparently  in  the  same  quantity  as  in  the 


TITRATION    OF   THE    COMPLEMENT. 


137 


control  tube,  and  form  there  a  large  deposit,  a  trace  of  hemolysis  or  almost 
o  would  be  the  terms  used  in  reporting  the  results.  Tube  7  after  two  hours 
showed  incomplete  hemolysis,  i.e.,  compared  with  control  III  it  was  noticeably 
clearer,  but  not  completely  transparent.  After  twenty-four  hours  there  was 
a  small  mass  of  undissolved  red  blood  cells  at  the  bottom  of  the  test  tube  and 
above  it  a  deep  red  fluid  which  was  only  slightly  different  from  that  in  the 
tubes  where  the  erythrocytes  were  completely  dissolved.  If  this  sediment 
should  become  so  small  that  on  shaking  only  a  cloudy  turbidity  is  produced, 
the  result  would  correspond  to  the  designations  "very  small  sediment," 
"  occasional  erythrocytes  at  the  bottom  of  the  tube,"  or  "  almost  complete 
hemolysis." 

In  the  tubes  containing  inactive  hemolysin  without  complement  (control  i,  and  in 
complement  binding  reactions)  hemagglutination  can  occur  because  the  agglutinins 
which  also  exist  in  the  serum  become  active.  Hemagglutination  is  recognized  by  the 
fact  that  on  shaking  the  sediment,  the  erythrocytes  are  not  equally  distributed,  but  remain 
in  clumps  or  strings  and  soon  sink  to  the  bottom  again. 

For  many  purposes  it  is  desirable  to  titrate  the  complement 

Titrationofthe  content  of  a  serum.     The  method  is  the  same  as  that  used 

Complement,  in  hemolysin  titration,  only  with  the  difference  that  a  fixed 

amount  of  hemolysin  and  varying  quantities  of  complement 
are  employed. 


Antigen. 

Amboceptor. 

Complement. 

N  p.          Result 
^aVL       after  two 
solutlonv      hours. 

I. 

2. 

3- 
4- 

i  c.c.  5%  of  sheep's  blood  . 
i  c.c.  5%  of  sheep's  blood  . 
i  c.c.  5%  of  sheep's  blood  . 
i  c.c.  5%  of  sheep's  blood  . 
c.c.  5%  of  sheep's  blood  . 
c.c.  5%  of  sheep's  blood  . 

c.c.  hemolysin  dil.  1:1000.  . 
c.c.  hemolysin  dil.  1:1000.  . 
c.c.  hemolysin  dil.  1:1000.  . 
c.c.  hemolysin  dil.  1:1000.  . 
c.c.  hemolysin  dil.  1:1000.  . 
c.c.  hemolysin  dil.  1:1000.  . 

T  c.c.     :io     (=  .01). 
0.8  c.c.    no    (  =  0.08). 
0.6  c.c.    :io    (=0.06). 
0.4  c.c.    :io    (  =  0.04). 
0.2  c.c.    :io    (=0.02). 
i.oc.c.    :ioo(  =  o.oi). 

2  .0  c.c.    complete. 
2.2.  c.c     complete. 
2.4  c.c.     complete. 
2.6  c.c.     complete. 
2.8  c.c.    Incomplete. 

2.OC.C.                   O 

8. 
9- 

c.c.  5%  of  sheep's  blood  . 
c.c.  5%  of  sheep's  blood  . 
c.c.  5%  of  sheep's  blood  . 

i  c.c.  hemolysin  dil.  1:1000.  . 

— 

i  c.c.  1:10    (  =  0.1)  ..  . 

3.0  c.c.  i          o 
3.0  c.c.            o 
4.0  c.c.             o 

The  titer  of  this  complement  when  employed  with  a  hemolysin  of  i/iooo  strength 
and  allowed  to  stay  in  the  incubator  for  two  hours  would  be  0.04. 

The  complement  content  of  the  serum  of  a  healthy  guinea-pig  is  fairly 
constant.  During  illness  the  titer  usually  is  decreased.  Among  healthy 
people  the  complement  titer  shows  marked  individual  variations. 

For  hemolysis  a  definite  quantitative  relationship  between  hemolysin 
and  complement  is  necessary. 

<*  On  the  basis  of  the  two  titrations  outlined  above,  it  has  been  estimated 
that  at  least  0.04  c.c.  complement  is  necessary  to  activate  o.ooi  c.c.  of 
hemolysin.  If  less  complement  is  used  with  the  same  amount  of  hemolysin, 
hemolysis  does  not  occur  or  else  it  is  incomplete.  If  the  quantity  of  hemo- 


138  BACTERIOLYSINS  AND   HEMOLYSINS. 

lysin  is  increased  for  instance  threefold,  then  it  will  be  found  that  0.02  c.c. 
of  complement  suffices  to  produce  hemolysis.  Vice  versa  with  an  excess  of 
complement  the  hemolysin  titer  of  o.ooi  c.c.  may  be  reduced.  However, 
there  are  narrow  limits  to  this  mutual  compensatory  action. 

Cytotoxins,  Cytolysins. 

The  hemolysin  bodies  are  characteristic  and  important  members  of  a 
general  class  of  substances  known  as  cytotoxins,  especially  investigated  by 
Metschnikoff  and  his  co-workers. 

Just  as  the  immunization  with  erythrocytes  led  to  the  production  of 
lytic  amboceptors  which  in  connection  with  complement  destroyed  and 
dissolved  their  antigens,  so  in  a  similar  manner,  various  substances  more  or 
less  specific  for  their  antigens  have  been  produced  through  immunization 
with  leucocytes  "Leucocidin;"  with  lierve  tissue,  "neurotoxin,"  with 
spermatozoa,  "spermatoxin,"  and  kidney  tissue,  "nephrotoxin."  The 
proof  of  their  action,  particularly  of  neuro-,  nephro-,  and  hepatotoxin  is 
not  simple.  As  all  these  cytotoxic  sera  have  at  the  same  time  a  hemolytic 
action,  it  is  not  easy  to  decide  to  what  extent  the  changes  in  the  organs 
observed  after  the  injection  of  the  cytotoxic  substances  are  dependent  upon 
the  action  of  hemolysins.  It  must  further  be  taken  into  consideration  that 
none  of  these  sera  are  absolutely  specific  for  the  organ  in  question.  This  is 
not  surprising,  inasmuch  as  there  are  widespread  common  group  charac- 
teristics (common  receptors)  among  the  different  organs  serving  as  antigens, 
and  only  very  few  groups  of  a  specific  nature.  The  hopes  which,  at  the 
beginning,  were  placed  upon  the  study  of  cytotoxins  particularly  with  the 
expectation  that  they  would  tend  to  become  diagnostic  and  therapeutic 
methods  for  the  treatment  of  malignant  tumors,  have  as  yet  been  unrealized. 
The  entire  field  of  cytotoxins  urgently  requires  further  investigation. 


CHAPTER  XIII. 
THE  METHOD  OF  COMPLEMENT  FIXATION. 

Its  principle,  antituberculin,  Ehrlich's  side-chain  theory,  serum  diagnosis  of  syphilis, 
and  diseases  caused  by  animal  parasites. 

It  has  already  been  demonstrated  that  neither  bacteriolysis 

The  Question  nor  hemolysis  can  take  place  without  the  presence  of  comple- 

of  Multipli-    ment.     The  question  therefore  arises  whether  this  complement 

city  of  Com-  is  the  same  in  both  of  these  reactions  or  whether  normal  serum 

plements.     possesses  different  complements.     In  order  to  solve  this,  a 

number  of  very  complicated  experiments  have  been  carried 
out  by  Ehrlich  and  Morgenroth,  Metschnikoff  and  Bordet  and  Gengou. 
Ehrlich  and  Morgenroth  endeavored  to  show  that  not  only  do  the  comple- 
ments of  different  animals  of  the  same  class  vary,  but  that  numerous  com- 
plements exist  within  one  individual  serum  (conception  of  the  multiplicity  of 
complements).  Metschnikoff  believed  that  each  serum  contained  at  least 
two  complements,  the  microcytase  and  the  macrocytase,  thus  enlisting  the 
supporters  of  a  dualistic  theory.  Bordet  and  his  school,  on  the  other  hand, 
although  agreeing  with  the  idea  that  the  complement  varies  in  different 
animals,  deny  its  multiplicity  and  contend  that  any  given  serum  contains 
but  one  alexin,  or  complement — the  theory  of  unity  of  complement.  It 
would  be  superfluous  to  cite  all  the  experimental  data  supporting  these 
opinions,  but  nevertheless  a  review  of  the  classical  experiment  of  Bordet  and 
Gengou  which  corroborated  the  existence  of  only  one  complement,  thus 
offering  the  fundamental  principle  for  the  establishment  of  the  most  impor- 
tant method  of  serum  diagnosis,  namely,  complement  fixation,  would  not  be 
out  of  place. 

Bordet  and  Gengou  mixed  in  a  test-tube   typhoid  bacteria 

The  Principles  (antigen),  inactivated  typhoid  immune  serum  (amboceptor) 

of  Comple-    and  normal  serum  (complement).     Union  of  the  bacteria  and 

ment  Fixation,  immune  serum  first  took  place  followed  by  absorption  of,  and 

coalescence  with,  the  bacteriolytic  complement  contained  in  the 
normal  serum.  As  a  result,  bacteriolysis  occurred  and  the  bacteriolytic  com- 
plement was  used  up  during  this  process.  Bordet  and  Gengou  reasoned 
that  if  the  bacteriolytic  and  hemolytic  complements  were  identical,  then  in 
the  above  mixture  of  typhoid  bacteria,  immune  serum  and  normal  serum, 
the  hemolytic  as  well  as  bacteriolytic  complement  should  be  absent,  while 


140  THE   METHOD    OF    COMPLEMENT   FIXATION. 

if  the  plurality  of  complement  exists,  the  hemolytic  complement  should 
still  be  present.  Accordingly,  after  a  certain  interval,  washed  erythrocytes 
and  inactivated  homologous  immune  serum  were  added  and  hemolysis  looked 
for.  No  hemolysis  took  place,  thereby  attesting  to  the  fact  that  the  bacteria 
in  the  first  part  of  the  test  had  "fixed"-  ("held  in  check")  not  only  the  bac- 
teriolytic  but  also  the  hemolytic  complement.  Bordet  and  Gengou  there- 
upon named  this  test  "complement  fixation"  or  "complement  binding" 
(La  fixation  d'alexine). 

With  the  aid  of  this  experiment  Bordet  and  Gengou  were  able  to  prove  a  number  of 
theoretically  important  points.  They  demonstrated  among  other  things  that  absorption 
of  complement  was  not  always  necessarily  accompanied  by  bacteriolysis.  For  example,  the 
anthrax  and  pest  bacteria  when  mixed  with  their  respective  homologous  immune  sera 
show  no  or  only  very  incomplete  bacteriolysis.  The  erroneous  conclusion  thus  reached 
to  the  effect  that  these  sera  contained  no  amboceptors,  was  disproved  by  Bordet  and 
Gengou,  who  demonstrated  that,  i.  these  sera  contained  amboceptors  in  spite  of  the 
absence  of  bacteriolysis,  2.  the  complement  was  absorbed,  although  no  bacteriolysis  took 
place. 

During  the  process  of  immunization,  amboceptors  were  found  far  more  frequently 
than  bacteriolysins.  These  two  terms  must  not  be  considered  as  synonymous. 

Amboceptor  signifies  a  more  generic  term,  and  one  must  differentiate  between  amboceptors 
of  cytolytic  and  non-lytic  properties.  Whether  the  difference  here  really  depends  upon  the 
different  nature  of  the  amboceptor,  or  upon  the  construction  and  constitution  of  the 
antigen,  is  not  solved. 

The  fixation  of  the  complement,  precedes  the  act  of  bacteriolysis.  The  important 
requirement  for  the  fixation  is  an  antigen  which  has  been  sensitized  by  the  attachment 
of  the  amboceptor,  thus  increasing  the  affinity  toward  the  haptophore  group  of  the  com- 
plement. Antigen  alone,  or  even  amboceptor  alone,  cannot  or  perhaps  only  very  slightly 
bind  the  complement.  Whether  the  zymotoxic  (energy)  group  of  the  complement 
manifests  its  activity  (bacteriolysis)  or  not  (absence  of  bacteriolysis)  is  materially  in- 
different for  the  complement  fixation. 

Through  complement  fixation,  as  introduced  by  Bordet  and 
Complement   Gengou,  one  *s  enabled  to  prove  the  presence  of  specific  anti- 
Fixation  as    bodies  when  the  antigen  is  known  or  reversely,  an  unknown 
a  Method     antigen  provided  the  specific  antibody  is  given.     This  method 
of  Serum      of  serum  diagnosis  can  be  widely  employed,  as  the  majority  of 
Diagnosis,     bacteria  and  immune  sera  (with  the  exception  of  pure  antitoxic 
sera)1  when  mixed  homologously,  give  a  positive  reaction— 
the  absence  of  hemolysis,  proving  the  absorption  of  complement  by  the  union 
of  the  antigen  and  its  specific  amboceptor.     This  reaction  is  strongly  specific. 
If  bacteria  are  mixed  with  an  inactive  heterologous  immune  serum,  or  with 
a  heated  normal  one,  not  in  concentrated  form  (normal  amboceptor),  and 
complement  is  added,  the  latter  will  not  be  fixed  but  remains  to  be  taken  up 
by  the  subsequently  added  red  blood  cells,  and  its  immune  serum,  causing 
hemolysis.     Hemolysis  indicates  that  the  mixed  bacteria  and  serum  are  not 

1  Even  antitoxic  sera  are  said  by  Nicolle  to  give  complement  fixation  reactions. 


COMPLEMENT   FIXATION   TESTS. 


141 


homologous,  as  the  complement  is  left  free,  and  given  a  chance  to  unite  with 
the  added  erythrocytes  and  hemolytic  amboceptor.  In  the  case  where  the 
bacteria  are  known,  e.g.,  typhoid  bacilli,  the  occurrence  of  hemolysis  indicates 
that  the  examined  serum  contains  no  typhoid  amboceptors.  If  the  serum 
is  known  (e.g.,  meningococcus  serum)  the  occurrence  of  hemolysis  proves 
that  the  bacteria  under  examination  are  not  meningococci.  The  absence  of 
hemolysis,  will  in  the  first  case  point  out  that  the  unknown  serum  contains 
typhoid  amboceptors,  i.e.,  is  a  typhoid  serum;  while  in  the  second  case  the 


Typhoid  bacilli  (antigen) 

Inactive   typhoid   serum    (typhoid 
amboceptor) 

Complement 


Hemolysin    (hemolytic    ambo- 
ceptor) 

Sheep's  red  blood  cells  (2nd  antigen) 
Result:     Due  to  the  union  of  complement 
with  the  complex  typhoid  bacillus  +  the 
typhoid  amboceptor.     Hemolysis  did  not 
take  place. 


Typhoid 
bacilli 

0 

V7       Blood  cell 

v 

Typhoid 
amboceptor 

A 

V 

Hemolytic 
amboceptor 

A 

Complement          / 

m 

FIG.  15. 


Typhoid  bacilli  (antigen) 
Inactive  cholera  serum  (cholera  ambo- 
ceptor) 

Complement 
Hemolysin 

Sheep's  red  blood  cells 
Result:     The  complement  unites  with  the 
hemolysin    and    the   sheep's    red    blood 
cells  thus  producing  hemolysis. 


Typhoid 
bacilli 


Cholera 
amboceptor 


FIG.  16. 


\  /     Red  blood  cell 

VI 


Hemolytic 
amboceptor 


Complement 


absence  of  hemolysis  would  bear  definite  evidence  in  favor  of  meningococci. 
The  accompanying  figures,  15  and  1 6,  represent  schematically,  the  positive 
and  negative  complement  fixation  test. 

Gengou  further  showed  that  not  only  cellular  antigens  can  stimulate  the 
formation  of  amboceptors,  but  that  during  the  course  of  immunization  with 
proteids  in  solution  (milk,  serum,  etc.),  complement  binding  amboceptors  are 
also  formed  in  addition  to  the  precipitins.  Citron  has  therefore  proposed  the 
term  "antigenophile,"  to  designate  the  "  cytophile"  group  of  the  amboceptor. 

Widal  and  Lesourd,  were  the  first  to  make  practical  application  of  the 
complement  fixation  property.  They  found  that  the  Bordet-Gengou  reaction 
could  be  obtained  far  more  frequently  and  earlier  with  the  serum  of  typhoid 


142  THE    METHOD    OF    COMPLEMENT    FIXATION. 

patients  than  the  agglutination  test.     Nevertheless,  this  entire  complement 
fixation  method  remained  unheeded  for  several  years. 

Moreschi  (at  Pfeiffer's  institute),  while  conducting  some  theoretical  studies  concerning 
the  nature  of  anticomplements,  i.e.,  such  substances  which  tend  to  neutralize  the  action 
of  complements,  discovered  anew,  that  by  the  mixture  of  a  soluble  proteid  with  its  anti- 
proteid  serum  the  existing  complement  disappeared.  This,  as  has  been  seen,  can  be 
explained  by  the  presence  within  the  immune  serum,  of  bodies  similar  to  Gengou's 
amboceptors.  Moreschi,  however,  stated  that  the  complement  disappeared  because  it 
was  thrown  to  the  bottom  mechanically,  by  the  occurrence  of  precipitation.  Such  a 
physical  explanation  for  the  complement  fixation  reaction  lead  a  number  of  authorities 
to  the  belief  that  the  positive  Bordet-Gengou  reaction  was  in  reality  no  amboceptor  ac- 
tion, but  a  result  of  a  similar  precipitation  process.  Wassermann  and  Bruck,  Liefmann, 
Wassermann  and  Citron,  and  later  on  Moreschi  himself  realized  that  this  physical  ex- 
planation was  incorrect,  inasmuch  as  complement  fixation  took  place  even  if  all  preci- 
pitation was  prevented  by  heat  or  other  influences.  Furthermore,  complement  binding 
of  an  unspecific  nature  can  be  produced  by  the  mixture  of  glycogen  or  peptone  with 
serum,  a  procedure  wherein  surely  no  precipitation  plays  any  part.  Finally  Moreschi 
showed  that  there  were  strongly  precipitating  sera  which  nevertheless  did  not  exhibit  the 
Bordet-Gengou  phenomenon. 

Thus  was  definitely  established  that  the  complement  fixation  was 
entirely  independent  of  either  bacteriolysis  or  precipitation. 

Following  Moreschi' s  researches,  Neisser  and  Sachs  continued  Gengou's 
studies  and  advised  this  demonstration  of  the  proteid  amboceptors  as  a 
control  to  the  precipitation  method  for  the  differentiation  of  proteids.  Its 
action  is  so  much  finer,  and  more  delicate  than  the  precipitin  test  that  even 
the  minutest  traces  of  proteid  can  be  recognized. 

With  the  encouraging  results  of  Neisser  and  Sachs  in  mind,  Wassermann 
attempted  by  the  use  of  highly  immune  antibacterial  serum  to  discover  any 
soluble  bacterial  proteids  which  may  exist  in  the  blood,  derived  from  the 
respective  bacteria  invading  the  organism  at  the  onset  of  an  infection. 
Practical  application  proved  that  not  enough  of  these  proteids  existed  free 
in  the  circulation,  but  that  they  were  probably  bound  by  the  tissue  cells. 

Wassermann  and  Bruck  then  employed  the  complement  fixation  test 
with  the  idea  of  demonstrating  the  existence  of  the  respective  antigens  in  the 
diseased  organs.     Tuberculous  glands   and   lungs  served  as  material  for 
this  experiment.     They  were  able  to  obtain  complement  fixation  when  an 
extract  of  tuberculous  organs  as  antigens  was  mixed  with  a  tuberculous 
serum  (manufactured  by  the  Hochst  Farbwerke).     If  instead  of  the  lat- 
ter, the  serum  from  tuberculous  individuals  was  substituted,  no  positive 
complement   fixation   reaction   was   obtained.     On   the   other   hand,   the 
reaction  was  given  if  the  human  tuberculous  serum  employed 
Antituber-     came  from  an  individual  who  had  received  therapeutic  inocu- 
culin.        lations  of  tuberculin.     In  other  words,  the  serum  of  treated 
individuals   contained,    in   contrast   to   the   untreated   ones, 
amboceptors  against  a  soluble  tuberculous  substance  also  present  in  the 


TUBERCULIN    THEORIES.  143 

extract  of  tuberculous  glands.  Wassermann  and  Bruck  identified  this 
substance  as  tuberculin,  because  the  sera  of  the  treated  individuals  gave  the 
same  positive  results  if  a  solution  of  old  or  new  tuberculin  was  used  instead 
of  the  extract  of  tuberculous  organs.  Thus,  the  latter  contained  tuberculin 
while  the  sera  of  the  tuberculin-treated  individuals  contained  amboceptors 
designated  by  Wassermann  and  Bruck  as  "antituberculin."  The  name 
antituberculin  has  not  been  a  well  chosen  one,  because  it  creates  the  impres- 
sion among  many  as  being  an  antitoxin.  It  is  better  to  speak  of  it  as  anti- 
tuberculin  amboceptors. 

Since,  according  to  Wassermann  and  Bruck  these  antituberculin  ambo- 
ceptors were  not  supposed  to  be  formed  spontaneously  in  tuberculous  indi- 
viduals, but  only  in  those  treated  with  tuberculin,  their  demonstration 
could  be  of  no  apparent  diagnostic  value.  On  the  other  hand,  their  exist- 
ence greatly  furthered  the  understanding  of  Koch's  tuberculin  reaction,  as 
most  tuberculous  individuals  who  had  antituberculin  amboceptors  in  their 
serum  did  not  respond  to  the  subcutaneous  injection  of  tuberculin. 

Wassermann  and  Bruck,  moreover,  showed  that  a  mixture  of  tuberculin 
with  an  extract  from  tuberculous  organs  bound  complement.  From  this 
they  concluded  that  the  extract  likewise  contains  antituberculin  amboceptors. 
Thus  reasoning  they  developed  their  tuberculin  theory. 

The  difference  in  the  reaction  observed  in  a  normal  and  tuberculous 

Tuberculin    individual  after  inoculation  of  tuberculin,  can  be  fully  explained  by  the 

Theory  of     presence  of  antituberculin  amboceptors  in  the  tuberculous  focus.     By 

Wassermann  virtue  of  their  specific  affinity,  the  amboceptors  attract  the  injected 

and  Bruck.     tuberculin   toward  them.     The  tuberculin    and    antituberculin    unite, 

and  absorb  the  complement  from  the  circulating  blood  stream,  since  the 

complementophile  group  of  the  amboceptor  is  free  and  unbound.     By  virtue  of  the  fresh 

complement  which  is  an  actively  lytic  ferment,  and  the  attracted  leucocytes,  a  partial 

destruction  and  casting  off  of  the  tuberculous  focus  results.     Upon  this  depends  the 

therapeutic   effect  of   the  tuberculin.     During  a  prolonged  treatment  with  tuberculin, 

the  body  produces  an  excess  of  antituberculin  amboceptors  so  that  finally  some  appear 

free  within  the  blood  serum.     When  this  is  the  case  the  tuberculous  organism  loses  its 

power  to  react  toward  tuberculin,  as  the  latter  is  neutralized  in  the  blood-stream  at  a 

point  away  from  the  local  focus.     No  therapeutic  effect  is  any  longer  obtained  from  the 

tuberculin  injections,  so  that  they  can,  for  a  time,  be  suspended.     The  aim  of  tuberculin 

therapy  should  be  to  work  with  small  doses  so  that  only  a  focal  reaction  is  obtained  and 

the  hyperproduction  of  antituberculin  amboceptors  be  postponed  as  long  as  possible. 

Numerous  exceptions  were  at  once  taken  to  this  theory  and  its  experimental  data, 
the  most  important  of  which  can  here  be  mentioned. 

Weil  and  Nakayama  disagreed  with  the  proof  of  the  existence  of  "anti- 
"Summier-     tuberculin"  in  the  organ  extracts,  on  the  basis  that  Wassermann  had 
ung's  Ein-       overlooked  the  effect  of  a  summation  of  antigen.     This  is  best  explained 
wand"  (Ex-    as  follows:     Complement  is  bound  not  only  by  antigen  +  amboceptor, 
ception  taken  but  also  by  large  doses  of  antigen  itself  dependent  upon  the  normally 
on  Ground  of    present  amboceptors  existing  in_the  serum  employed  for  complement. 
Summation  of] 
Antigen.) 


144 


THE   METHOD    OF    COMPLEMENT   FIXATION. 


Old  tuberculin. 

Complement. 

Erythrocytes. 

Hemolysin. 

Result. 

0.05 

O.I 

0.15 

O.I 
O.I 
O.I 

i  c.c.  5% 
i  c.c.  5% 
i  c.c.  5% 

Twice  the  hemolytic  titer. 
Twice  the  hemolytic  liter. 
Twice  the  hemolytic  titer. 

Hemolysis. 
Hemolysis. 
No  hemolysis. 

0.15  old  tuberculin  is  thus  sufficient  of  its  own  accord  to  bind  complement, 
their  experiment,  Wassermann  and  Bruck  found  that, 


In 


Tuber- 
culin. 

+  Extract  of 
tuberculous 
organs. 

Complement. 

Erythrocytes. 

Hemolysin. 

Result. 

O.I 
O    I 

O.I 

O.  I 
O    I 

i  c.c.  5% 

ICC     5% 

Twice  the  hemolytic  dose. 
Twice  the  hemolytic  dose 

Complement 
fixation. 
Hemolysis 

O.I 

O.  I 

i  c.c.  5% 

Twice  the  hemolytic    dose. 

Hemolysis. 

This,  however,  in  no  way  proves  the  existence  of  "antituberculin"  in  the  extract  of 
tuberculous  organs,  as  it  is  perfectly  possible  and  even  probable  that  o.i  of  the  organ 
extract  contains  0.05  c.c.  at  least  of  tubercle  bacillus  substance  (tuberculin)  which  when 
added  to  o.i  of  tuberculin  used  for  antigen,  is  sufficient  to  give  an  amount  of  tuberculin 
perfectly  capable,  as  has  been  seen,  of  binding  complement  by  its  own  activity. 

In  order  to  overcome  this  possibility  one  must  work  with  such  small 
but  at  the  same  time  maximum  amounts  of  antigen  and  antibodies,  that  at 
least  double  the  quantity  of  each  of  these  reagents  does  not,  of  its  own 
accord,  bind  complement.  For  tuberculin  this  is  estimated  as  follows: 


Tuberculin. 

Complement. 

Hemolysin. 

Erythrocyte. 

Result. 

O.2 

O.I 

2  X  Hemolytic  dose 

c.c.  5% 

No  hemolysis 

0.18 

o. 

2  X  Hemolytic  dose 

c.c.  5% 

No  hemolysis 

0.15 

o. 

2  X  Hemolytic  dose 

c.c.  5% 

No  hemolysis 

0.14 

o. 

2  X  Hemolytic  dose 

c.c.  5% 

Incomplete  hemolysis 

O.  12 

0. 

2  X  Hemolytic  dose 

c.c.  5% 

Complete  hemolysis 

O.I 

0. 

2  X  Hemolytic  dose 

c.c.  5% 

Complete  hemolysis 

Twelve-hundredths  is  the  maximum  non-binding  or  hemolytic  dose. 
For  the  complement  fixation  test  where  the  object  is  to  demonstrate  anti- 
tuberculin  amboceptors,  the  maximum  amount  of  antigen  to  be  used  is 
therefore  0.06  T.  or  one-half  of  the  maximum  non-binding  dose. 

In  the  same  way  the  hemolytic  dose,  and  the  antibody  containing  reagent, 
should  be  estimated. 


EXCEPTIONS    TO    COMPLEMENT   FIXATION. 


Organ  extract. 

Complement. 

Hemolysin. 

Erythrocyte. 

Result. 

O.  2 

0.  I 

2XHemolytic  dose 

i  c.c.  5% 

o 

o.  16 

O.I 

2  X  Hemoly  tic  dose 

i  c.c.  5% 

Complete 

hemolysis 

O.  I 

O.  I 

2  X  Hemoly  tic  dose 

i  c.c.  5% 

Complete 

hemolysis 

The  non-binding  dose  is  0.16.  The  amount,  however,  to  be  employed  in 
the  complement  fixation  test  must  be  0.08  c.c.  of  organ  extract. 

If  on  mixing  0.06  T.  and  0.08  extract,  complement  fixation  still  appears, 
then  this  summation  of  antigen  can  be  disregarded  and  an  antigen  antibody 
reaction  must  be  considered.  For,  even  granting  that  0.08  of  extract  does 
for  its  greater  part,  e.g.,  0.06  at  the  most,  contain  tuberculin,  then  this 
amount  +  0.06  of  the  tuberculin  in  the  antigen  only  makes  0.12  of  tuberculin, 
a  quantity  not  sufficient  to  fix  the  complement.  De  facto,  complement 
fixation  does  occur  when  the  above  test  is  carried  out  with  proper  dosage, 
so  that  most  probably  it  is  occasioned  by  the  biological  antigen  antibody 
reaction.  As  a  general  rule  for  all  complement  fixation  tests,  the  dose  of  each 
ingredient  employed  should  never  be  more  than  i  /2  of  its  maximum  complement 
non-binding  quantity. 

This  principle  was  not  definitely  established  at  the  time  of  Wassermann  and  Bruck's 
first  studies  so  that  experimental  proof  for  the  existence  of  antituberculin  amboceptors 
in  tuberculous  organs  has  not  been  corroborated. 

A  second  exception,  taken  by  Weil  and  Nakayama  as  well  as 

Other  by  Morgenroth  and  Rabinowitsch  relates  to  the  activity  of  the 
Exceptions,  complement  when  it  combines  with  tuberculin  and  anti- 
tuberculin. 

They  claim  that  by  this  union  the  complement's  lytic  function  is  entirely 
lost.  Morgenroth  and  Rabinowitsch  even  go  so  far  as  to  deny  the  existence 
of  antituberculin  in  the  blood  of  tuberculous  individuals. 

The  author  also  undertook  a  minute  study  of  this  question  and  came  to  the 
following  definite  conclusion. — There  are  some  tuberculous  individuals  who 
spontaneously  develop  antituberculin  amboceptors,  a  fact  to  be  expected 
because  it  has  for  a  long  time  been  known  that  on  and  off  tuberculin  can  be 
liberated  in  the  organism  of  tuberculous  individuals.  As  a  natural  conse- 
quence antibodies  will  be  formed,  and  most  probably  by  those  tissue  cells  in 
the  neighborhood  of  the  liberation  of  the  tuberculin,  i.e.,  the  focus  of  infection. 

Before  proceeding,  however,  to  the  author's  conception  of  the  tuberculin 
theory  it  is  necessary  to  review  Ehrlich's  principles  of  immunity  upon  which 
the  ideas  of  antibodies  and  their  specificity  are  based. 


146  THE   METHOD    OF   COMPLEMENT   FIXATION. 

Ehrlich's  idea  of  the  biological  structure  of  cells  is  that  they  consist 

Ehrlich's      of  two  parts,  a  central  functionating  radicle  ("Leistungskern")  upon 
Side  Chain     which  depends  the  specialized  activities  of  the  cells,  as  for  example,  a 

Theory.  glandular  or  nerve  cell,  and  a  multiplicity  of  side  chains  or  receptors 
(a  term  borrowed  from  the  chemistry  of  the  benzol  group),  by  means 
of  which  the  cell  enters  into  chemical  relation  with  food  and  other  substances  brought 
to  it  by  the  circulation.  These  receptors  are  exceedingly  numerous,  as  the  nutritive 
substances  upon  which  the  cell  depends  for  its  maintenance  are  very  varied.  Besides 
these  general  receptors  the  special  cells  also  have  different  and  special  side  chains;  then, 
too,  there  exist  very  great  quantitative  differences  among  the  latter;  and  finally  it  must  be 
added  that  the  selective  activity  of  the  cells  depends  upon  the  variability  of  these 
receptors. 

When  an  infection  occurs,  pathological  material  is  brought  to  the  cell  bodies  instead 
of  physiological  normal  substances.  Certain  of  these  poisonous  products  find  suitable 
receptors  in  all  of  the  cell  groups,  others  fit  only  into  distinct  groups  of  cells,  while  a 
third  class  are  not  taken  up  at  all.  This  is  strikingly  in  evidence,  for  the  organism 
which  possesses  no  receptors  for  any  of  the  pathological  agents,  cannot  assimilate  any 
deleterious  substances  and  is  therefore  immune.  Lack  of  amboceptors  is  therefore  a 
natural  form  of  immunity.  The  organism  having  only  a  special  group  of  cells  for  the 
reception  of  certain  pathological  matter,  will  make  use  of  these  cells  for  the  binding  and 
assimilation  of  the  toxic  material.  For  example,  the  nerve  cells  alone  have  receptors 
for  tetanospasmin;  no  matter  how  or  when  the  poison  is  introduced  into  the  organism 
the  nerve  cells  will  absorb  it.  As  this  toxin  is  poisonous  for  the  central  atom  group 
(Leistungskern)  of  the  nerve  cell,  the  latter  is  destroyed.  The  union  between  the  nerve 
cell  receptors  and  the  tetanospasmin  toxin  is  only  the  preliminary  act  for  the  cell  destruc- 
tion; the  actual  death  of  the  cell  being  caused  by  the  action  of  the  toxophore  group  of 
the  poison  upon  the  functional  radicle  of  the  cell.  If,  however,  such  receptive  side 
chains  are  possessed  not  only  by  the  brain  but  also  by  other  cells,  e.g.,  connective  tissue 
cells,  the  tetanospasmin  will  in  part  be  bound  by  the  latter.  The  toxophore  group  of 
the  toxin  does  not  have  any  harmful  effect  upon  the  functional  radicle  of  these  cells, 
and  thus  no  toxic  effects  will  be  incurred  by  the  union,  and  the  nerve  cells  remain 
unaffected. 

The  number  of  receptors  which  cells  possess  for  tetanospasmin,  for  example,  are  limited 
and  after  their  junction  with  the  teanospasmin,  are  rendered  useless  and  inactive.  By 
the  normal  reparative  mechanism  of  the  body,  new  receptors  are  generated.  This 
reparative  process  does  not  as  a  rule  stop  at  a  simple  replacement  of  lost  elements,  but 
according  to  the  hypothesis  of  Weigert  tends  to  overcompensation.  The  receptors 
eliminated  by  toxin  absorption  are  reproduced  in  an  excess  of  the  simple  physiological 
needs  of  the  cell.  Continuous  and  increasing  dosage  of  the  toxin  soon  leads  1o  such 
excessive  production  of  receptors  that  the  latter  find  no  more  room  to  be  attached  to  the 
cell,  but  are  cast  off  and  circulate  free  in  the  blood.  They  still,  however,  retain  their 
property  of  being  able  to  combine  with  tetanospasmin. 

If  such  an  organism  is  injected  with  tetanospasmin  the  latter  toxin  is  bound  by  the 
free  receptors  in  the  serum,  and  thus  the  respective  "sessile"  receptors  attached  to  the 
cells  are  precluded  from  coming  in  contact  with  the  poison.  Inasmuch  as  the  free 
receptors  possess  no  functional  radicle  which  can  be  injured,  the  toxin  remains  entirely 
innocuous  for  the  individual.  Such  protective  bodies  lend  to  the  organism  its  attained 
immunity  and  are  known  as  antitoxins.  Their  function  can  be  compared  to  lightning  rods. 

v.  Behring  well  expresses  their  action  when  he  states  that  the  same  elements  which 
attached  to  the  cells  render  the  body  susceptible  to  toxic  substances,  when  circulating 
freely  in  the  blood  serve  to  protect  it. 


EHRLICH  S    SIDE   CHAIN   THEORY.  147 

The  antibodies  against  toxins  and  ferments  are  of  the  simplest  form.  They  possess 
only  a  binding  group  which  has  an  affinity  toward  the  haptophore  group  of  the  toxins 
and  ferments.  They,  therefore,  belong  to  the  class  designated  by  Ehrlich  as  "hap- 
tines"  of  the  first  order. 

To  the  haptines  of  the  second  order  belong  the  agglutinins  and  precinitins.  They 
possess  besides  a  haptophore  group  also  an  agglutinophore  or  percipitinophore  group 
by  virtue  of  which  agglutination  or  precipitation  takes  place. 

Belonging  to  the  haptines  of  the  third  order  are  the  class  of  amboceptors  which  have 
in  addition  to  the  haptophore  group  also  a  complementophile  group  for  their  union 
with  the  complement. 

These  hypotheses  of  Ehrlich  greatly  simplify  the  explanation  of  many  serum  eactions 
as  well  as  many  of  the  phenomena  associated  with  the  action  of  tuberculin.  In  all 
probability  the  healthy  cells  which  exist  in  the  tuberculous  focus  and  which  are  capable 
of  reaction,  produce  the  antituberculin.  Christian  and  Rosenblatt  offered  experimental 
evidences  for  this  statement.  They  demonstrated  that  tuberculous  guinea-pigs  in  whom 
antituberculin  was  produced  by  tuberculin  injections,  showed  a  diminution  of  anti- 
tuberculin  in  the  blood  when  tuberculous  glands  were  removed  by  operation. 

The  antituberculin  production  by  the  cells  is  a  transitory  action  arising  only  when 
tuberculin  has  spontaneously  or  artificially  reached  the  circulation.  Following  this 
stage  of  activity  there  comes  a  period  of  quiescence  during  which  no  free  antituberculin 
can  be  found  in  the  serum.  The  cells,  however,  are  supplied  with  a  great  many  more 
sessile  receptors  than  usually;  they  possess  a  higher  affinity  toward  tuberculin  and 
produce  antituberculin  much  more  readily  than  normal  cells. 

This  also  explains  why  the  smallest  amounts  of  tuberculin  produce  a  reaction  in 
tuberculous  and  not  in  the  normal  individuals.  In  the  former,  the  cells  in  the  zone 
surrounding  the  tuberculous  focus  are  abundantly  supplied  with  receptors,  so  that  on 
the  injection  of  tuberculin,  its  action  appears  almost  concentrated  at  this  point.  Occa- 
sionally the  sessile  receptors  are  relatively  scarce  and  the  first  injection  excites  no 
reaction.  '  By  the  time  of  the  second  or  third  inoculation  these  sessile  amboceptors 
have  so  increased  that  a  positive  reaction  is  apparent  when  the  same  or  even  a  smaller 
dose  is  injected.  This  phenomenon  of  increased  sessile  receptors  explains  the  reappear- 
ance of  subsided,  subcutaneous,  cutaneous,  or  ophthalmo  reactions  after  renewed 
injections  of  tuberculin. 

To  recapitulate  the  biological  phenomena  associated  with  a  positive 
tuberculin  reaction,  it  may  be  said  that  the  tubercle  bacilli,  or  portions  of 
their  body  substances  existing  in  the  infected  focus,  stimulate  the  adjacent 
cells  to  produce  a  great  number  of  sessile  receptors.  When  the  tuberculin 
is  injected  for  the  first  time,  these  sessile  receptors  at  once  take  up  the  tuber- 
culin and  as  a  result,  the  production  of  antituberculin  in  the  focus  is  further 
stimulated.  Every  production  of  antibodies,  is,  if  the  stimulant  be  strong 
enough,  associated  with  fever;  in  this  very  regard,  however,  Wright  as  well  as 
Pfeiffer  and  Friedberger  showed  that  if  the  smallest  doses  of  antigen  are 
employed,  antibody  production  continues  without  any  rise  in  temperature. 
Fever  in  a  tuberculin  reaction  is  therefore  not  a  necessary  manifestation  of  a 
positive  tuberculin  reaction,  although  it  generally  is  present.  The  enlarged 
number  of  sessile  antituberculin  receptors  augments  the  affinity  of  the  cells 
toward  the  tuberculin,  and  the  second,  third  and  succeeding  inoculations 
bring  about  a  focal  reaction  (i.e.,  antituberculin  production)  much  more 


148  THE   METHOD    OF    COMPLEMENT   FIXATION. 

easily.  Finally  the  antituberculin  receptors  become  so  numerous  that  they 
are  detached  from  the  cells  and  become  free  receptors.  This  period,  however, 
is  only  transitory,  as  is  corroborated  by  the  difficulty  connected  with  the 
demonstration  of  these  antibodies  in  the  focus.  This  free  antituberculin 
combines  with  the  tuberculin  (spontaneously  formed  or  injected)  and  attracts 
the  complement,  or  the  complement  producing  phagocytes.  Uncombined 
complement  has  no  effect  on  the  tissues.  It  is  different,  however,  with  the 
phagocytes.  These  can  without  any  additional  help  act  directly  upon  the 
infected  focus.  If  the  tuberculin  treatment  is  continued,  a  period  arises 
during  which  the  antituberculin  bodies  are  so  greatly  accumulated  in  the 
local  focus  that  they  ultimately  escape  into  the  blood  stream.  This  freely 
circulating  antituberculin  neutralizes  any  freshly  injected  tuberculin,  so 
that  such  patients  become  refractory  even  against  the  largest  amounts  of  it. 
(Tuberculin  immunity.)  Tuberculin  immunity  is  not,  however,  in  all  cases 
to  be  identified  with  a  strong  antituberculin  content  in  the  serum.  For 
example,  it  is  very  difficult  to  stimulate  antituberculin  by  treatment  with 
S.  B.  E.,  although  by  its  use  an  immunity  against  B.  E.  is  easily  attained. 

In  former  times  a  negative  tuberculin  reaction  after  a  prolonged  treatment  was 
stamped  as  a  cure  of  the  tuberculosis,  a  fact  obviously  incorrect;  for,  no  matter  how 
successful  the  tuberculin  therapy  may  be,  it  cannot  be  considered  as  a  complete  curative 
procedure. 

The  appearance  of  antituberculin  in  the  general  circulation  is  interpreted  in  a  double 
light.  Wassermann  and  Bruck  advised  that  it  was  best  to  avoid  its  appearance,  because 
by  its  presence  here  the  tuberculin  is  neutralized  without  ever  reaching  the  focus  where 
it  is  required.  On  the  other  hand,  it  may  be  considered  a  protective  element  in  that  it 
binds  any  tuberculin  which  may  spontaneously  be  formed  in  the  system.  In  general, 
that  method  should  be  adopted  which  makes  the  subject  non-susceptible  to  the  largest  doses  of 
tuberculin.  In  practice  it  was  found  that  those  patients  having  the  greatest  amounts  of 
antituberculin  in  their  serum,  generally  offered  a  better  prognosis  than  the  others. 

Recent  experiments  of  the  author  seemed  to  show  that  in  certain  cases  serum  con- 
taining antituberculin  can  raise  the  susceptibility  for  tuberculin.  Thus  tuberculous 
guinea-pigs  injected  with  a  mixture  of  tuberculin,  antituberculin  and  complement  in  pro- 
portionate dosage,  died  in  several  hours,  while  animals  of  the  same  kind  receiving  only 
tuberculin  or  tuberculin  +  antituberculin  remained  alive. 

i  The  experiences  gained  by  the  employment  of  the  complement 

Serum       fixation  test  in  tuberculosis,  lead  to  its  application  in  the  study 

Diagnosis  of  of  syphilis.     The  difficulties  in  this  disease  were  greater,  inas- 

Syphilis.      much  as  there  were  no  bacteria  or  preparations  like  tuberculin 

which  could  be  used  as  antigen. 

Syphilitic  organ  extracts  were  employed  instead,  with  the  idea  that  these  would 
contain  the  specific  virus.  The  serum  of  monkeys  previously  immunized  with  such 
extracts  when  mixed  in  vitro  with  the  latter,  gave  complement  fixation.  This  experi- 
ment is  not,  however,  conclusive  as  the  positive  reaction  may  be  due  to  anti-human 
proteid  amboceptors  produced  at  the  same  time  by  the  injection  of  the  human  serum 
contained  in  the  organ  extract.  The  experiment  was  changed  and  the  syphilitic  organ 


SERUM   DIAGNOSIS    OF    SYPHILIS. 


149 


extracts  from  apes  were  used  so  as  to  exclude  the  error.  Even  in  this  way  complement 
fixation  was  attained.  Later  on  it  was  found  unnecessary  to  inject  the  monkeys  with 
the  extracts  since  after  ordinary  infection  their  serum  would  give  complement  fixation. 
In  this  manner  it  was  almost  definitely  established  firstly,  that  these  extracts  contained 
a  substance  specific  for  syphilis  which  could  with  most  probability  be  considered  a 
luetic  antigen,  and  secondly  that  infected  apes  possessed  antibodies  against  this  antigen. 

The  next  step  was  to  try  the  reaction  in  man.  The  first  experiments  of 
Wassermann,  Neisser,  Bruck  and  Schucht  did  not  give  the  hoped  for 
returns.  Although  the  reaction  was  obtained  with  human  serum,  the  per- 
centage of  positive  results  was  so  small  (see  next  chart)  that  its  practical 
value  as  a  means  of  diagnosis  offered  no  great  help.  Only  in  general 
paralysis  did  the  expectation  seem  promising.  In  about  80  per  cent,  of  all 
cases  Wassermann  and  Plaut  were  able  to  demonstrate  the  luetic  antibodies 
in  the  cerebrospinal  fluid. 

Schiitze's  experiments  in  tabes  led  him  to  the  same  findings.  Citron  has  obtained  a 
much  smaller  percentage  of  positive  reacting  cerebrospinal  fluids  in  tabes. 

As  it  seemed  that  the  means  of  diagnosis  was  not  to  be  established  by  the 
demonstration  of  the  syphilitic  antibody,  Neisser  and  Bruck  believed  that 
better  results  may  possibly  be  achieved  by  the  discovery  of  the  luetic  antigen 
in  the  serum  through  complement  fixation. 

This  attempt  too  was  unsuccessful.  No  antigen  could  be  found,  but  the  extracts  of 
red  blood  cells  from  syphilitic  individuals  when  mixed  with  the  serum  of  highly  immunized 
monkeys  gave  a  positive  complement  fixation.  Neisser  and  his  co-workers  concluded 
therefrom  that  the  erythrocyte  extract  contained  the  luetic  antigen.  Citron  soon  demon- 
strated that  the  extracts  of  normal  individuals  gave  a  similar  reaction  and  what  was  more 
important,  that  this  so-called  blood  antigen  existed  in  the  blood  entirely  uninfluenced 
by  mercurial  treatment.  Since  these  experiments,  not  much  importance  has  been  attached 
to  this  reaction. 

Meanwhile  the  author  working  at  the  Kraus  clinic  proved  by  a  large 
series  of  experiments  that  luetic  antibodies  were  present  in  almost  all  cases 
of  lues.  The  reaction  is  dependent  upon  two  rules. 

The  First. — The  longer  the  syphilis  virus  has  acted  upon  the  organism  and 
the  more  numerous  its  recurrent  manifestations  have  been,  the  more  fre- 
quently will  a  positive  reaction  be  obtained  and  the  stronger  will  the  anti- 
body content  of  the  serum  be. 

The  Second.— The  sooner  a  proper  mercury  therapy  is  instituted,  the 
more  often  it  is  repeated,  and  the  shorter  the  interval  since  the  last  treatment, 
the  smaller  will  the  antibody  content  of  the  serum  be  and  the  greater  the 
possibility  of  a  negative  reaction. 

These  points  were  soon  corroborated  by  numerous  other  workers  in  the 
field,  so  that  at  the.  present  day,  they  can  be  taken  as  absolute  facts.  The 
following  chart  will  explain  some  of  the  statements  aforementioned. 


THE   METHOD    OF   COMPLEMENT   FIXATION. 


First  period. 

Second  period. 

• 

Wassermann,  Neisser, 
Bruck  &  Schucht. 

Wassermann  and 
Plaut. 

Marie  and  Levaditi. 

Morgenroth  and 
Stertz. 

Schiitze. 

Citron. 

e 

C/2 

1 

E 

PQ 

Fleischmann. 

o 

'C 

1 
1 

i 
& 

1 

CJ 

§ 

tf 

I 

g 

"8 

<N 

3rd.  report. 
(with  Blaschko.) 

Lues  I 

8 

% 

% 

% 

% 

% 

% 

oo 

4.8    2 

IOO 

68 

C2    6 

% 

Lues  II 

26  7 

yw 
98 

T-  °  •  *" 
7Q 

Q7 

Q-2 

o*  •** 

IOO 

02.8 

with 

*\j  .  f 

V 

/  V 

vo 

vo 

V       w 

symp- 

toms. 

without 

14.6 

80 

20 

64 

75-6 

46 

symp- 

/ 0 

toms 

("early 

latent") 

Lues  III 

77    ^ 

•  74. 

with 

21.6 

/  /  •  J 

/  *» 

I 

cy  .4 

98 

IOO 

O2  .  2 

88.9 

symp- 

o/ •  i 

y 

V 

ww     y 

toms 

without 

ii  i 

1:7 

2O.  2 

42 

36.8 

77  .  c 

symp- 

0 / 

o 

o  /    o 

toms 

("late 

latent") 

Pro- 

(80) 

(75) 

(100) 

IOO 

gressive 

.... 

\t***t 

(spinal 

\l  3J 

(spinal 

\             / 

(spinal 

paraly- 

fluid) 

fluid) 

fluid) 

88 

sis 

Tabes 

(66) 

86.6 

7O 

60 

dorsalis 

\         / 

(spinal 

(22) 

/  V 

fluid) 

(spinal 

fluid) 

As  has  been  repeatedly  remarked,  specificity  is  the  important  element  in 
every  biological  reaction.  The  reaction  known  after  the  discoverer  as  the 
Wassermann  Reaction,  can  also  be  performed  if  instead  of  the  extract  from 
luetic  organs,  an  alcoholic  extract  of  certain  normal  organs  or  certain  lipoid 
substances  is  substituted  as  antigen.  Seligmann  too,  was  able  to  obtain 
complement  fixation  by  pure  chemical  reactions.  Consequently,  numerous 
authorities  expressed  the  opinion  that  the  Wassermann  Test  was  non- 
specific and  that  it  does  not  at  all  represent  an  antigen  antibody  interaction. 

There  is  no  doubt,  however,  that  this  exceptional  view  is  incorrect.  It  is  true  that  the 
real  syphilitic  antigen  is  unknown,  but  most  probably  it  is  neither  the  pure  spirochaetes 


WASSERMANN   REACTION.  151 

nor  a  pure  lipoid  substance.  The  author  has  expressed  the  hypothesis  to  the  effect  that 
the  antibody  producing  antigen  is  a  toxolipoid.  This  explains  the  fact  why  pure  lipoids 
can  stimulate  no  antibodies,  but  can  at  the  same  time  react  with  luetic  antibodies  in 
vitro. 

The  accompanying  diagram  (Fig.  17)  explains  this  hypothesis.  In 
order  to  answer  the  objections  raised  against  this  theory,  the  author  has 
proposed  the  indifferent  term  of  "Lues- 

reagine"  for  the  luetic  antibodies  as  long  f  V7    Syphilis  virus 

as  their  biological  structure  is  unknown.   Lues-Antigen  j  vW 

Independent    of    the     question     of  U/    Lipoid  aecithinj 


"biological  specificity,"  the  Wassermann 


reaction  must  also  be  considered  in  the 

Amboceptor 

light  of  a  "  clinical  specificity."     From 


Lipoidophile  group 


Complementophile 
group 


this  standpoint  it  fulfills  its  demands. 
With  only  few  exceptions,  it  can  be  con- 
sidered absolutely  specific  for  lues.  /  \  Complement 

The  well  established  exceptions  are,  fram- 
bcesis,  trypanosomiasis,  leprosy,  malaria,  scarlet,  pIG    I^> 

febris  recurrens.     The  reactions  obtained  here 

are  similar,  but  not  the  same  as  those  obtained  in  syphilis.  In  leprosy  the  point  of 
difference  is  seen  in  that  the  reaction  can  also  be  performed  with  tuberculin  as  antigen; 
in  scarlet  the  reaction  appears  only  in  a  small  percentage  of  cases  and  not  with  all  luetic 
extracts.  Furthermore,  it  disappears  at  the  latest  three  months  after  the  infection,  usually 
much  sooner.  As  for  trypanosomiasis  and  malaria  convincing  data  are  still  too  few. 

These  diseases  excluded,  a  positive  Wassermann  reaction  can  be  taken 
as  certain  proof  for  the  existence  of  lues.  Whether  such  a  test  is  indicative 
of  a  by-gone  infection  or  whether  it  means  that  an  active  process  is  still 
going  on  at  the  time  of  its  obtention  has  been  for  a  long  time  a  subject  of 
discussion.  The  author  is  of  the  firm  opinion  that  the  demonstration  of  the 
"lues  reagine"  means  active  Iws.  The  reasons  for  this  belief  are  as  follows: 

1.  The  almost  constant  presence  of  the  reaction  in  all  cases  of  manifest 
lues  excepting  primary  lesions.     During  this  stage  it  is  entirely  absent  or 
only  partly  detected.     It  appears,  however,  later  on. 

2.  The  practically  assured  existence  of  the  reaction  with  a  recurrence  of 
symptoms  even  if  before  that  the  reaction  was  negative. 

3.  The  possibility  of  influencing  a  positive  reaction  so  that  it  becomes 
negative,  by  the  use  of  mercury.     The  latter  holds  true  also  for  those  cases 
which  show  no  symptoms  and  are  therefore  incorrectly  designated  as  latent 
syphilis.     It  has  been  proven  that  such  are  in  reality  by  no  means  latent, 
but  have  an  active  process  at  some  point  escaping  detection,  as  the  aorta. 
Only  cases  of  a  nature  which  have  no  symptoms  and  a  negative  reaction 
should  be  considered  as  latent  syphilis,  those  however  with  no  symptoms, 
but  a  positive  reaction  as  belonging  to  the  class  of  active  lues. 

4.  The  evidence  that  apparently  healthy  individuals,  but  with  a  positive 


152  THE   METHOD    OF    COMPLEMENT   FIXATION. 

reaction,  have  infected  others  or  have  all  of  a  sudden  developed  tertiary 
or  postluetic  manifestations,  tabes,  paralysis,  diseases  of  the  aorta,  etc. 

An  objection  has  frequently  been  raised,  that  in  spite  of  existing  disease,  the  reaction 
has  been  found  negative.  If  the  statistics  covering  the  largest  number  of  cases  are 
studied,  it  will  be  seen  that  such  instances  are  rare.  Few  exceptions  are  discovered  in 
every  biological  reaction,  especially  one  which  is  complicated  and  where  five  different 
ingredients  come  into  play;  even  in  the  immunization  of  animals  differences  will  be  found 
in  that  some  produce  a  highly  agglutinating  or  precipitating,  etc.,  serum,  while  others 
will  show  few  or  even  no  antibodies.  Individual  differences  are  prevalent  to  such  extent 
that  exceptions  to  the  rule  must  be  taken  for  granted.  Fortunately,  a  negative  reaction 
in  existing  lues  is  so  rare,  that  for  practical  purposes  its  possibility  may  be  overlooked, 
at  least,  with  reservation. 

As  a  general  rule,  antibodies  persist  in  an  organism  for  a  certain  time  past  infection, 
when  the  individual  has  become  perfectly  well.  Exceptions,  to  the  effect  that  it  may 
be  possible  for  a  positive  Wassermann  reaction  to  similarly  signify  a  past  infection  or  a 
state  of  immunity,  have  been  raised.  But  it  must  be  said  that  immunity  in  syphilis  is  a 
condition  thus  far  unproven  and  almost  unknown.  All  symptoms  previously  attributed  to 
such  an  immunity  can  more  easily  be  explained  in  the  light  of  a  continuation  of  the 
disease.  As  for  the  "lues  reagine"  remaining  after  the  cure  of  the  infection,  undoubtedly 
this  phenomenon  is  possible.  The  analogy  with  other  diseases  seems  lost,  however, 
when  one  considers  that  the  syphilitic  reaction  is  discovered  thirty  or  forty  years  after  an 
infection,  while  antibodies  in  general  persist  for  weeks,  months  or  at  the  most  for  several 
years,  following  an  infection.  Still  it  may  be  possible  that  the  syphilis  "reagine"  is  char- 
acterized by  the  difficulty  with  which  it  is  excreted  and  by  the  tendency  of  the  cells  when 
once  stimulated  to  produce  antibodies  to  continue  to  do  so.  The  influence  of  mercury, 
however,  demonstrates  that  this  phenomenon  is  closely  allied  to  similar  actions  exhibited 
by  the  class  of  bacteria.  If  a  patient  whose  serum  gives  a  positive  reaction  is  subjected 
to  mercurial  treatment,  the  reaction  becomes  negative  in  several  weeks.  The  mercury 
has  destroyed  the  stimulant  or  irritant  which  has  led  the  cells  to  the  production  of  anti- 
bodies. If  this  stimulant  is  excluded,  the  "lues  reagine"  disappears  from  the  blood  just 
as  bacterial  antibodies  disappear  after  the  bacteria  have  been  eradicated.  Thus  there 
is  no  basis  for  attributing  to  the  luetic  antibodies  any  exceptional  properties. 

The  fact  that  mercury  leads  to  an  alteration  in  the  reaction, 

Citron's      prompted  the  author  to  employ  the  Wassermann  test  as  a 

"Biological    guide  to  the  biological  mercurial  treatment.     The  aim  was  not 

Mercurial       only  to  cause  a  disappearance  of  all  manifestations,  but  to  obtain 

Treatment."   a  negative  reaction.     It  soon  appeared  that  a  negative  reaction 

once  obtained  did  not  necessarily  remain  such.     As  soon  as  a 

recurrence  set  in  the  reaction  became  positive  again;  in  fact,  the  reaction 

also  reappeared  without  a  return  of  symptoms.     In  the  latter  case  such  a 

return  alone  was  regarded  as  a  fresh  manifestation  of  a  reactivation  process 

and  an  indication  for  treatment.     It  became  advisable  therefore,  to  repeat 

the  test  at  definite  intervals  and  depend  upon  the  return  of  the  reaction  for 

further  treatment.     This  basis  of  therapy,  which  at  first  met  with  marked 

opposition,  has  recently  won  many  followers. 

The  experiments  of  Boas  in  Copenhagen  are  especially  instructive 
from  this  point  of  view. 


153 

He  examined  eighty-two  patients  with  secondary  syphilis  before  and  after  mercurial 
therapy.  All  gave  positive  reactions  before  the  treatment;  after  it,  seventy-six  gave  no 
reaction,  six  retained  the  positive  reactions;  one  of  the  six  did  not  return  for  observation. 
Of  the  remaining  five,  all  had  a  return  of  symptoms  within  one  month  after  cessation  of 
the  mercury,  while  of  the  seventy-six  only  three  returned  with  a  recurrence.  Boas  next 
made  observations  of  sixty-five  patients  who  were  in  the  first  three  years  of  their  infection, 
but  who  gave  a  negative  Wassermann  after  the  treatment.  In  sixty-two  cases,  a  positive 
reaction  reappeared  after  one  to  two  months,  eight  of  these  having  at  the  same  time  a 
recurrence  of  symptoms;  of  the  remaining  fifty-four,  nineteen  were  not  treated.  They 
all  showed  a  return  of  symptoms,  but  only  one  and  a  half  months  after  the  appearance 
of  the  positive  Wassermann.  Thus  if  the  scheme  of  the  chronic  intermittent  mercurial 
therapy  of  Neisser  and  Fournier  were  followed,  these  patients  would  begin  to  get  treatment 
one  and  a  half  months  after  the  active  lues  had  again  started,  as  shown  by  the  positive 
Wassermann  reaction.  Of  the  remaining  thirty-five  cases  all  began  treatment  when  the 
Wassermann  test  became  positive.  None  of  these  had  any  return  of  symptoms  during  the 
following  period  of  observation  (three  to  five  months). 

The  experiments  of  Boas  show  distinctly  the  advantages  of  the  mercurial 
therapy  when  based  upon  the  biological  reaction  instead  of  upon  the  sche- 
matic, symptomatic,  chronic,  intermittent  treatment  of  Fournier  and  Neisser. 

At  the  present  day,  when  the  spirochaetes  can  be  so  readily  found  in  the 
primary  lesion  of  syphilis,  the  biological  mercurial  treatment  should  be 
undertaken  in  the  earliest  stage.  It  is  possible  even  to  begin  at  a  time  when 
the  serum  reaction  is  still  negative,  but  after  the  spirochaete  had  been 
demonstrated.  The  most  ideal  cases  are  those  in  which  treatment  is 
instituted  so  early  that  they  never  develop  a  positive  Wassermann. 

Naturally  the  statement  made  that  mercurial  treatment  should  be  continued  until 
the  reaction  becomes  negative  may  be  limited  by  certain  contra-indications  in  the  general 
condition  of  the  patient  which  may  arise.  This  must  always  be  considered.  Especial 
difficulty  to  attain  a  negative  reaction  is  encountered  in  those  cases  where  the  lues  has 
persisted  for  many  years. 

It  must  be  borne  in  mind  that  the  luetic  infection  does  not  always  present 
the  typical  clinical  picture  ascribed  to  it  in  the  text-books.  The  "Lues 
asymptomatica, "  that  is,  the  lues  apparently  presenting  no  symptoms,  is  by 
no  means  rare.  To-day  one  must  not  wait  until  the  syphilitic  patient  comes 
to  the  physician,  but  it  is  the  duty  of  the  latter  to  look  for  the  evidence  of  syphilis 
among  those  related  to  or  associated  with  infected  persons.  If  one  proceeds 
in  such  a  systematic  method  it  will  be  found  that  the  mothers  of  syphilitic 
children,  so  frequently  regarded  as  immune,  are  in  reality  not  so.  In  such 
cases,  without  any  clinical  evidence  of  syphilis,  the  Wassermann  reaction  is 
positive  in  about  56  to  75  per  cent. 

This  question  becomes  of  utmost  importance  in  the  prevention  of  lues. 
For  example  the  obligatory  examination  of  the  serum  of  wet  nurses  has 
shown  that  of  all  such  applicants  at  the  Dresden  Infant  Asylum  10  per  cent, 
gave  a  positive  reaction  (Rietschels).  On  further  study  it  was  ascertained 


154  THE   METHOD    OF   COMPLEMENT   FIXATION. 

that  75  per  cent,  of  the  children  of  these  apparently  healthy  women  gave 

luetic  manifestations  immediately  or  shortly  after  birth. 
Serum  Diag-  *n  c^ose  association  with  the  serum  diagnosis  of  syphilis,  com- 
noJFs  of  Dis-  plement  fixation  has  been  employed  as  a  means  for  the  diagno- 
eases  Caused  sis  of  conditions  caused  by  the  animal  parasites  and  especially 
by  Animal  by  the  echinococcus.  In  the  serum  of  patients  suffering 
'es*  from  these  infections,  substances  are  found  closely  allied  to 

the  "lues  reagine."     They  bind  complement  with  an  antigen  consisting  of 

an  extract  of  the  respective  worms  or  hydatid  fluid. 

Ghedini,  Weinberg  and  Parvu  and  others  have  found  that  in  most  cases  of  echino- 
coccus disease,  the  reaction  is  positive.  If  by  operation  the  cyst  is  only  incised  the  re- 
action becomes  stronger  or  in  few  cases  appears  positive  for  the  first  time.  After  com- 
plete excision  of  the  cyst,  the  reaction  disappears.  According  to  Parvu  and  Laubry,  a 
positive  test  is  found  in  the  spinal  fluid  only  when  the  echinococcus  cysts  have  invaded  the 
brain. 

Ghedini  described  similar  findings,  caused  by  the  ascaris,  ankylostoma, 
etc. 


CHAPTER  XIV. 
THE  TECHNIQUE  OF  COMPLEMENT  FIXATION. 

Original  method  of  Bordet-Gengou.  Wassermann-Bruck's  modification.  Technique 
of  serum  diagnosis  for  syphilis.  Echinococcus  disease.  Differentiation  of  proteids  ac- 
cording to  Neisser-Sachs. 

I.  The  Original  Method  of  Bordet-Gengou. 

a.  The  antigen  consists  of  bacteria  grown  upon  agar  for  twenty-four 
hours  and  then  suspended  in  physiological  salt  solution  to  make  a  rather 
concentrated  emulsion. 

For  typhoid  bacteria  Bordet  and  Gengou  take  5  c.c.  of  salt  solution  to  each  culture  of 
bacteria. 

For  tubercle  bacilli  80  mg.  of  the  bacteria  are  suspended  in  i  c.c.  of  salt  solution. 

b.  The  serum  containing  the  antibody  is  heated  for  one-half  hour  at 
56°  C.  to  destroy  the  complement. 

c.  As  complement,  the  fresh  serum  of  a  normal  animal  or  human  being 
is  used. 

d.  The  hemolysin  is  produced  by  the  inactivated  serum  of  a  rabbit 
that  had  been  immunized  against  sheep's  or  goat's  erythrocytes,  or  the 
serum  of  a  guinea  pig  injected  with  rabbit's  red  blood  cells. 

e.  The  respective  red  blood  corpuscles  are  washed,  to  free  them  of 
their  complement  containing  serum. 

A  definite  amount  of  bacterial  suspension  is  mixed  with  varying  amounts  of  inactivated 
immune  serum  and  a  proportional  amount  of  complement  is  added.  These  three  ingre- 
dients are  mixed  and  allowed  to  remain  at  room  temperature  for  four  to  five  hours. 
During  this  time  the  complement  is  fixed  if  the  antigen  and  antibody  are  of  a  homologous 
nature.  In  order  to  see  whether  this  union  has  taken  place  or  not,  hemolysin  and 
erythrocytes  are  added  in  a  mixture  thus  prepared:  2  c.c.  of  inactivated  hemolysin  + 
twenty  drops  of  washed  blood  cells  are  mixed  and  allowed  to  remain  together  for  about 
fifteen  minutes  so  that  the  erythrocytes  are  sensitized,  i.e.,  united  with  the  hemolytic 
amboceptor.  Of  this  mixture  each  tube  receives  o.i  to  0.2  c.c.  If  the  complement 
has  not  become  fixed,  hemolysis  occurs  in  several  minutes.  If  the  complement  has 
become  so,  hemolysis  does  not  occur;  since,  however,  the  hemolysin  also  contains 
hemagglutinin,  the  erythrocytes  are  agglutinated  and  sink  to  the  bottom  of  the  tubes. 
As  control  tests,  Bordet  and  Gengou  considered  the  following  very  necessary: 
i.  Bacterial  suspension  +  inactivated  normal  serum  (instead  of  immune  serum) 
+  complement  (five  hours)  +  hemolysin  +  blood.  Hemolysis  must  occur,  as  the 
normal  serum  does  not  contain  enough  amboceptors  to  unite  with  the  bacterial  suspen- 
sion and  consequently  complement  remains  unbound. 

155 


156 


THE   TECHNIQUE    OF    COMPLEMENT   FIXATION. 


2.  Inactivated  immune  serum   +   complement  (five  hours)   +  hemolysin  +  blood. 
Hemolysis  results. 

3.  Inactivated  normal  serum  +  complement  (five  hours)  +  hemolysin  +  blood.    He- 
molysis. 

4.  Antigen  +  inactivated  immune  serum  +  hemolysin  +  blood.    No  hemolysis,  as 
complement  is  absent. 

5.  Antigen   +  inactivated  normal  serum  (five  hours)    +  hemolysin  +  blood.     No 
hemolysis,  as  complement  is  absent. 

The  following  is  the  chart  of  the  first  complement  fixation  test  as  originally  performed 
by  Bordet  and  Gengou  in  1901  in  which  pest  antibodies  were  demonstrated  in  the  serum 
of  an  immunized  horse. 


Antigen. 

Antibodies. 

Complement. 

Hemolysin  and 
erythrocytes. 

Result. 

I 

o  .  4     pest     bacilli 
emulsion. 

i  .  2   inactive  pest 
serum  (horse). 

o  .  2     guinea-pig's 
serum. 

2  drops  of  rabbit's 
blood  sensitized. 

o 

2 

0.4  pest  bacilli 
emulsion. 

i  .  2  inactive  nor- 
mal    serum 
(horse). 

o  .  2  guinea-pig's 
serum. 

2  drops  of  rabbit's 
blood  sensitized. 

Complete 
hemolysis. 

•2 

i  2  inactive  pest 

o  2     Eruinea-Difif's 

2  drops  of  rabbit's 

Complete 

serum  (horse). 

serum. 

blood  sensitized. 

hemolysis. 

4 

i  2  inactive  nor- 

o 2     guinea-pig's 

2  drops  of  rabbit's 

Complete 

mal  serum  (horse)  . 

serum. 

blood  sensitized. 

hemolysis. 

5 

o  .  4     pest     bacilli 

i  .  2  inactive  pest 

2  drops  of  rabbit's 

o 

emulsion. 

(horse). 

blood  sensitized. 

6 

0.4     pest     bacilli 

i  2  inactive  nor- 

2 drops  of  rabbit's 

o 

emulsion. 

mal    serum 
(horse). 

blood  sensitized. 

Employing  this  method,  Bordet  and  Gengou  found  positive  results  with  the  following 
combinations: 

1.  Pest  bacilli  +  pest  horse's  serum  +  guinea-pig  complement  +  guinea  pig  hemolysin 
+  rabbit's  blood. 

2.  Anthrax  vaccine  +  guinea-pig  immune  serum  +  guinea-pig  complement  +  guinea-pig 
hemolysin  +  rabbit's  blood. 

3.  Typhoid  bacilli  -1-  guinea-pig  immune  serum  +  guinea-pig  complement  +  guinea- 
pig  hemolysin  +  rabbit's  blood. 

4.  Coli  bacilli  +  guinea-pig  immune  serum  +  guinea  pig  complement  +  guinea-pig 
hemolysin -I- rabbit's  blood. 


WASSERMANN-BRUCK'S  MODIFICATION.  157 

5.  Typhoid  bacilli  +  human  convalescent  serum  +  human  complement  +  guinea-pig 
hemolysin+  rabbit's  blood. 

6.  Killed   tubercle   bacilli  4- guinea-pig   immune   serum  +  guinea-pig   complement  + 
rabbit's  hemolysin  +  goat's  blood  or  sheep's  blood. 

7.  Whooping     cough    bacilli  +  patient's    serum -(-guinea-pig's    complement -I- rabbit 
hemolysin  +  goat's  or  sheep's  blood. 

8.  Meningococci  +  human  convalescent's  serum  +  human  complement  +  guinea-pig's 
hemolysin  +  rabbit's  blood  (Cohen). 

In  addition  Widal  and  Lesourd  on  examination  of  sixty-one  typhoid 
cases  found  fifty-eight  with  a  positive  reaction.  Foix  and  Mallein  examined 
twelve  cases  of  scarlet  and  obtained  a  positive  result  in  ten  cases  when  the 
streptococcus  grown  from  a  scarlet  angina  was  used  as  antigen.  Antibodies 
were  found  on  the  fourth  day.  These  results  were  confirmed  by  Schleissner. 

II.  Wassermann-B ruck's  Modification. 

a.  Antigen. — Instead   of   entire   bacteria,    only   bacterial   extracts   are 
employed.     These  are  made  in  the  same  manner  as  the  artificial  aggressins. 

For  typhoid  bacteria  Leuchs  advises  that  the  bacterial  suspension  should  first  be 
killed  for  twenty-four  hours  at  60°  C.  and  then  shaken  for  two  days.  In  tuberculosis 
good  results  are  obtained  by  using  Koch's  preparation  of  old  and  new  tuberculin. 

The  bacterial  extracts  when  very  fresh  contain  a  great  deal  of  precipitino- 
gen  which  diminishes  in  several  days  and  finally  disappears.  Its  presence 
does  not  disturb  complement  fixation.  The  bacterial  extracts  must  be  well 
protected  from  light  and  kept  in  the  cold. 

After  the  extract  has  stood  for  some  time  a  sediment  forms;  under  no  circumstance 
should  this  be  disturbed  or  shaken.  The  required  amount  of  antigen  should  be  carefully 
poured  off,  and  not  pipetted  off.  Just  as  soon  as  the  required  amount  is  obtained,  the 
extract  should  be  returned  to  the  ice-box. 

b.  The  antiserum  is  inactivated  by  heating,  even  if  the  serum  is  old  and 
contains  very  little  or  no  complement. 

Old,  non-heated  serum  is  often  antihemolytic.  Temperatures  over  60°  C.  should  be 
strictly  guarded  against  as  the  amboceptors  may  be  destroyed.  Heating  for  a  period 
longer  than  one-half  hour  may  make  a  serum  anticomplementary,  i-.e.,  bind  comple- 
ment. Sera  containing  bile  at  times  prevent  hemolysis.  Chylous  sera  obtained 
during  the  period  of  digestion  and  milky  sera  seen  in  nursing  women  do  not  differ  from 
the  normal. 

Exudates,  transudates,  and  spinal  fluids  are  treated  like  sera. 

c.  Complement  is  obtained  by  killing  a  guinea-pig  and  using  its  serum 
while  fresh.     The  serum  preserved  in  "Frigo"  is,  according  to  Sterns,  not 
reliable. 

d.  Hemolysin  is  represented  by  the  inactivated  serum  of  a  rabbit  that 
has  been  immunized  against  sheep's  red  blood  cells. 

e.  The  twice  washed  sheep's  red  blood  cells  are  used  as  erythrocytes. 


'58 


THE   TECHNIQUE    OF   COMPLEMENT   FIXATION. 


These  five  substances  are  placed  into  the  test  tubes  in  the  following  order:  antigen, 
inactivated  antiserum,  complement;  they  are  thoroughly  mixed  by  shaking  and  placed 
into  the  incubator  for  one  hour  in  order  to  hasten  their  union.  After  this  interval  the 
inactivated  hemolysin  and  the  red  blood  cells  are  added  as  indicator.  The  mixtures 
are  again  returned  to  the  incubator  to  promote  hemolysis.  Like  in  all  biological  reactions, 
the  quantitative  relationship  of  these  various  ingredients  determines  to  a  great  extent 
the  final  result  of  the  complement  fixation  test.  As  for  antigen  and  antibody  the  experi- 
ments of  Weil  and  Nakayama  must  be  considered;  these  are  to  the  effect  that  only 
one-half  of  the  maximum  non-hemolytic  dose  of  each  ingredient  is  employed.  With 
this  point  in  view,  preliminary  tests  determining  the  proper  dosage  of  each  must  be 
performed. 

The  amount  of  complement  used  is  always  constant.  In  Wassermann's  laboratory 
i  c.c.  of  the  dilution  1:10  represents  the  quantity  chosen.  Of  the  hemolysin  the  two 
fold  or  three  fold  titre  dose  is  taken  and  of  the  erythrocytes  i  c.c.  of  a  5  per  cent,  sus- 
pension in  normal  saline  solution  suffices.  Each  of  the  five  elements  is  diluted  with  saline 
to  make  up  i  c.c.  so  that  at  the  completion  of  the  test  all  the  tubes  contain  5  c.c.  Quite 
a  difference  arises  if  an  individual  test  is  performed  with  a  constant  quantity  of  serum 
and  diminishing  doses  of  bacterial  extract  or  reversely.  Important  tests  should  be 
carried  out  by  both  methods.  The  necessary  controls  are: 

1.  The  double  dose  of  antigen  +  complement  +  hemolysin  +  blood,  to  prove  that  the 
dose  employed  in  the  test  is  correct  (Weil  and  Nakayama). 

2.  The  double  quantity  of  serum  +  complement  +  hemolysin  +  blood,  to  prove  that  the 
dose  of  serum  employed  is  correct  (Weil  and  Nakayama). 

3.  The  "system  control";  blood  +  complement-!- one-half   amount  of  hemolysin,  to 
show  that  the  test  was  performed  with  double  the  hemolytic  dose. 

4.  Blood  +  salt  solution,  to  prove  that  the  salt  solution  is  isotonic. 

In  addition,  it  is  advisable  to  repeat  the  test  with  inactivated  normal  serum  substituted 
for  the  immune  serum  and  another  with  a  foreign  instead  of  a  homologous  antigen. 
These  controls  assure  beyond  doubt  the  specificity  of  the  reaction. 
The  accompanying  chart  represents  schematically  all  that  has  been  discussed. 

Titration  of  a  Meningococcus  Serum  Obtained  from  the  Horse, 
a.  Diminishing  Quantities  of  Antigen. 


Antigen. 

Antibodies. 

Complement. 

Hemolysin. 

Erythrocytes. 

Results. 

, 

0.25  c.c. 

o  .  i  c.c.  inactive 

O.I  C.C. 

0.002  c.c.  in- 

i c.c.  5% 

Meningococcus 

immune  serum. 

guinea-pig's 

active  rabbit's 

sheep's  blood 

0 

extract 

serum. 

(sheep)  serum. 

O.I  C.C. 

" 

" 

" 

" 

0 

0.05  c.c. 

a 

u 

u 

u 

0 

O.OI  C.C. 

a 

u 

* 

u 

0 

0.005  c.c. 

• 

u 

u 

« 

Incomplete 

O.OOI  C.C. 

• 

u 

u 

" 

Incomplete 

0.0005  c.c. 

a 

" 

u 

u 

Complete 

I    O  C  C 

u 

u 

• 

Incomplete 

O    ^  C  C 

a 

u 

a 

Complete 

o  25  c  c 

u 

a. 

u 

Complete 

WASSERMANN-BRUCK'S  MODIFICATION. 


Antigen. 

Antibodies. 

Complement. 

Hemolysin. 

Erythrocytes. 

Results. 

Inactive  immune 

serum. 
0.4  c.c. 

O    2  C.C. 

a 

a 

« 

u. 

u 

Incomplete 
Complete 

O.I  C.C. 

u 

u 

u 

Complete 

u 

O    OOI  C  C 

u 

Complete 

u 

o 

Meningococcus 
extract. 
0.25  c.c. 

O.I  C.C. 

Inactive  normal 
horse's  serum. 

O.I  C.C. 
O.I  C.C. 
O    2  C  C 

u 

« 
U 

O.OO2  C.C. 

« 
a 

u 

u 
a 

Almost 
Complete 

Complete 
Complete 

Staphylococcus 
extract. 
0.25  c.c. 

O.I  C.C. 

o.  s  c.c. 

Inactive  mening- 
ococcus  serum. 

O.I  C.C. 
O.I  C.C. 

u 

u 
u 

H 
U 

u 

u 

u 
u 

Complete 

Complete 
Complete 

The  titre  of  the  meningococcus  serum  is  o.ooi  c.c.  of  antigen.  Since  i.o  c.c.  of 
antigen  binds  o.i  complement  and  0.5  c.c.  does  not  interfere  with  hemolysis,  the 
maximum  dose  of  antigen  which  may  be  used  for  the  trial  is  0.25  c.c. 

Inasmuch  as  0.4  c.c.  of  the  inactivated  serum  binds  a  part  of  the  complement,  and 
o .  2  does  not  at  all  interfere  with  hemolysis,  the  maximum  dose  of  serum  to  be  employed 
is  o.i  c.c. 

The  positive  reaction  must  be  attributed  to  the  interaction  between  antigen  and 
antibody,  as  hardly  any  complement  fixation  takes  place  by  using  inactivated  normal 
serum  with  0.25  c.c.  of  antigen.  That  the  reaction  is  specific  is  shown  by  hemolysis 
occurring  when  the  homologous  antigen  is  substituted  by  a  Staphylococcus  extract. 

b.  Same  with  Diminishing  Amounts  of  Serum. 


Antigen. 

Antibodies. 

Complement. 

Hemolysin. 

Erythrocytes. 

Result. 

0.25  c.c.  menin- 

o.i c.c.  inactive 

O.I  C.C. 

0.002 

i  c.c.  5% 

0 

gococcus  extract 

meningococcus 

guinea-pig's 

sheep's  blood 

serum. 

serum. 

u 

0.05  c.c. 

a 

O.OO2 

a 

o 

u 

O.OI  C.C. 

u 

0.002 

a 

o 

u. 

0.005  c.c. 

« 

O.OO2 

* 

0 

u 

O.OOI  C.C. 

a 

O.002 

« 

Almost  o 

u 

0.0005  c-c- 

« 

O.OO2 

a 

Incomplete. 

u 

O.OOOI  C.C. 

« 

O.002 

u 

Complete. 

o  5  c  c  menin- 

u 

O.O02 

a 

Complete. 

gococcus  extract 

i6o 


THE   TECHNIQUE    OF    COMPLEMENT   FIXATION. 


Antigen. 

Antibodies. 

Complement. 

Hemolysin. 

Erythrocytes. 

Results. 

O    2  C  C. 

u 

O.OO2 

u 

Complete. 

u, 

O    OOI 

u 

Complete 

u 

o 

0.25  c.c.  raenin- 
gococcus  extract. 

u 

Inactive  normal 
horse's  serum. 

O.I  C.C. 

0.05  c.c. 

u 

0.002 
0.002 

u 
•  u 

Almost 
complete. 

Complete. 

0.2  c.c. 

u 

0.002 

u, 

Complete. 

0.25  c.c.  staphy- 
lococcus  extract. 

o  .  i  c.c.  inactive 
meningococcus 
serum. 

O.OO2 

u 

Complete. 

o   c  c  c  staphy- 

u 

O    OO2 

u 

Complete 

lococcus  extract. 

With  0.25  c.c.  of  antigen  the  titre  of  this  meningococcus  serum  is  0.0005  c.c.  of 
serum.  One  could  with  this  constant  quantity  of  serum,  and  varying  quantities  of 
antigen,  titrate  the  minimum  amount  of  antigen  necessary  for  complement  fixation.  It 
would  even  be  preferable  for  such  a  test  to  employ  0.005  c-c-  °f  the  serum,  as  this  amount 
surely  binds  no  complement.  If  such  a  titration  is  undertaken  it  will  be  found  that 
0.005  c-c-  °f  serum  with  0.05  c.c.  of  extract  can  bind  o.i  c.c.  of  complement. 

Similarly  the  antibodies  contained  in  the  blood  serum  or  spinal  fluid  of  a 
patient  can  be  determined  by  means  of  complement  fixation. 

If  it  is  desired  to  demonstrate  the  antigen  instead  of  antibody,  one  pro- 
ceeds as  follows: 

c.  Demonstration  of  meningococcus  antigen  in  the  spinal  fluid  of  a  patient 
with  a  possible  meningitis. 


Antigen. 

Antibodies. 

Complement. 

Hemolysin. 

Blood. 

Results. 

0.5  c.c.  active 

o.  i  c.c.  inactive 

O.I 

O.OO2 

i  c.c.  5% 

0 

spinal  fluid  from 

horse's  meningo- 

patient. 

coccus  serum. 

0.3  c.c. 

O.  I  C.C. 

0.  I 

0.002 

i  c.c.  5% 

Incomplete. 

O.I  C.C. 

O.I  C.C. 

0.  I 

0.002 

i  c.c.  5% 

Complete. 

O    2  C  C 

O    I 

O    OO2 

I  C.C,  5% 

Complete. 

I    O  C  C 

O    I 

O    OO2 

ICC    cr% 

Almost 

o  6  c.c 

O    I 

O.OO2 

i  c.c.  5% 

complete. 
Complete. 

WASSERMANN-BRUCK'S  MODIFICATION. 


161 


Antigen. 

Antibodies. 

Complement. 

Hemolysin. 

Erythrocytes. 

Results. 

0.  I 

O.OOI 

i  c.c.  5% 
i  c.c.  5% 
i  c.c.  5% 

i  c.c.  5% 

Complete, 
o 
Complete. 

Complete. 

0.5  c.c.  active 
normal  spinal 
fluid. 

I  .0  C.C. 

O.I   C.C. 

O.I 
O.I 

O.O02 
O.OO2 

0.5  c.c.  of  active 
spinal  fluid  from 
patient. 
0.3  c.c. 

o.i  c.c.  inactive 
normal  horse's 

serum. 

• 

O.I 
O.I 

O.O02 
0.002 

i  c.c.  5% 
i  c.c.  5% 

Almost 
complete. 

Complete. 

0.05  c.c.  menin- 
gococcus  extract. 

0.005  c-c-  inac- 
tive meningococ- 
cus  serum. 

O.I 

0.002 

i  c.c.  5% 

0 

Look  at 
previous 
test. 

The  spinal  fluid  contains  meningococcus  antigen  thus  proving  that  the  patient  is 
suffering  from  epidemic  cerebrospinal  meningitis.  Neither  the  double  amount  of  serum, 
a  double  amount  of  antigen,  a  mixture  of  normal  spinal  fluid  with  specific  serum,  nor 
normal  serum  with  the  specific  spinal  fluid  binds  complement.  Only  a  mixture  of  meningo- 
coccus extract  and  specific  serum  gives  complement  fixation. 

The  results  gotten  by  complement  fixation  greatly  depend  upon  the 
quantitative  relationship  of  the  various  ingredients.  The  affinity  toward 
the  complement  existing  between  the  antigen  and  amboceptor  on  the  one 
hand,  is  balanced  by  that  between  the  hemolysin  -h  blood  on  the  other. 
By  modifying  their  quantitative  proportions  different  results  maybe  obtained. 
If  for  example  the  strength  of  the  hemolysin  is  excessively  increased,  it  is 
possible  that  the  previously  bound  complement  is  again  detached  and  hemo- 
lysis  ensues.  Originally  the  results  were  read  after  the  mixtures  had 
remained  two  hours  in  the  incubator  and  twenty-four  hours  in  the  ice-box. 
At  the  present,  most  authorities  agree  to  read  the  results  at  a  time  when  the 
control  tubes  are  ready;  that  is  when  the  complement  is  bound  or  hemolysis 
has  been  completed  in  those  tubes  in  which  these  respective  phenomena 
should  occur. 

Wassermann's  modification  of  the  Bordet-Gengou  method  was  first 
practically  employed  for  the  titration  of  the  therapeutic  meningococcus 
serum.  One  cannot,  however,  correctly  judge  the  prophylactic  or  curative 
value  of  a  serum  by  its  antibody  content  as  they  do  not  run  hand  in  hand 
[R.  Kraus,  Garbat,  Citron.]  The  complement  fixation  method  was  also 
applied  by  Bruck  for  the  diagnosis  of  epidemic  meningitis;  by  Miiller  and 
Oppenheim  for  the  diagnosis  of  general  gonococcus  infections  (gonorrheal 
arthritis,  iridocyclitis,  etc.)  and  by  Hirschfeld,  Leuchs  and  Schone  and  others, 
in  typhoid — all  with  favorable  results. 


1 62  THE   TECHNIQUE    OF   COMPLEMENT   FIXATION. 

III.  Serum  Diagnosis  of  Syphilis.    , 

a.  Wassermann's  Technique. 

The  technique  of  this  reaction  as  carried  out  in  Wassermann's  laboratory 
is  practically  identical  with  that  just  described  for  the  diagnosis  of  bacterial 
infections.  The  preparation  of  the  antigen  varies  slightly. 

The  liver  obtained  from  a  syphilitic  fetus  is  weighed  and  cut  up  into  fine  pieces*. 
Four  times  its  weight  of  1/2  per  cent,  of  carbolic  solution  in  saline  is  added  and  the 
mixture  placed  into  a  brown  bottle  and  shaken  for  twenty-four  hours.  It  is  then  centrif  u 
galized  until  the  larger  liver  remnants  settle  to  the  bottom  and  a  somewhat  turbid 
fluid  remains  above.  The  latter  is  poured  off  into  a  brown  bottle  and  placed  into  the 
ice-box.  After  several  days  of  sedimentation,  the  fluid  assumes  a  yellowish-brown 
opalescence  and  can  now  be  used  as  a  luetic  antigen.  It  should  not  be  exposed  to  light 
and  heat,  should  not  be  shaken,  and  its  contents  should  not  be  pipetted  off,  but  care- 
fully poured  off  without  disturbance  to  the  sediment. 

By  titration  of  the  extract,  that  dose  is  determined  which  does  not  of 
itself  bind  complement.  Only  such  extracts  are  kept  which  in  the  dose  of 
0.4  c.c.  do  not  interfere  with  hemolysis. 

Control  tests  should  also  be  made  to  ascertain  whether  the  organ  extract 
has  any  tendency  of  its  own  to  hemolyse  red  blood  cells  without  the  presence 
of  complement  or  hemolysin. 

Not  every  luetic  extract  can  serve  as  antigen  for  complement  fixation. 
During  the  process  of  extraction  a  number  of  other  substances,  both  normal 
and  pathological,  may  be  drawn  from  the  luetic  liver  besides  that  agent 
necessary  for  the  Wassermann  test.  These  undesired  ingredients  may 
interfere  with  the  efficiency  of  the  extract.  For  this  reason  a  great  number 
of  known  positive  and  negative  sera  should  be  tested  with  each  new  extract, 
and  only  if  the  results  are  absolutely  correct  should  it  be  employed  as  antigen. 

In  the  early  work  of  Wassermann  the  antigen  was  described  as  deteriorating  very 
easily;  its  activity  would  either  be  entirely  destroyed  or  it  would  become  anticomple- 
mentary.  The  author  is  firmly  convinced  that  these  changes  are  brought  about  by 
careless  handling  of  the  extract  or  its  exposure  to  light.  If  properly  taken  care  of,  its 
activity  remains  constant. 

From  practical  experience,  it  has  been  found  that  extracts  which  must  be 
used  in  amounts  less  than  o.i  c.c.  are  as  a  general  rule  unsatisfactory. 
Similarly,  the  luetic  sera  are  most  active  when  doses  of  0.2  and  o.i  c.c.  are 
employed.  Amounts  greater  than  0.2  may  result  in  an  unspecific  reaction. 
The  most  favorable  combinations  are, 

0.2  c.c.  of  extract  +  0.2  c.c.  serum, 
o.i  c.c.  of  extract  +  o.i  c.c.  serum. 

The  accompanying  table  presents  the  titration  of  an  antigen  in  detail. 


SERUM   DIAGNOSIS    OF    SYPHILIS. 

a.  Preliminary  Test — Titration  of  the  Antigen. 


i63 


Antigen. 

Comple- 
ment. 

Hemolysin. 

Blood. 

Result. 

0.8  c.c.  luetic  extract 
0.6  c.c.  luetic  extract 
0.4  c.c.  luetic  extract 
0.2  c.c.  luetic  extract 

O.I 
O.I 
O.I 
O.I 

Twice  the  hemolytic  dose. 
Twice  the  hemolytic  dose. 
Twice  the  hemolytic  dose. 
Twice  the  hemolytic  dose. 

i  c.c.  5% 
i  c.c.  5% 
i  c.c.  5% 
i  c.c.  5% 

Incomplete  hemolysis. 

0 

Complete  hemolysis. 
Complete  hemolysis. 

o  8  c  c.  luetic  extract 

I  C.C.   S% 

Incomplete. 

o  6  c  c    luetic  extract 

i  c  c.  s% 

o 

0.4  c.c.  luetic  extract 

i  c.c.  5% 

o 

o  2  c  c    luetic  extract 

i  c.c.  <;% 

o 

The  test  proves  that  0.4  c.c.  of  extract  is  not  able  to  bind  o.i  c.c.  of 
complement.  That  0.8  c.c.  of  lues  extract  causes  only  an  incomplete  hemo- 
lysis, while  0.6  c.c.  produces  no  hemolysis  whatever,  is  explained  not  by  its 
lessened  tendency  of  binding  complement,  but  by  the  greater  amount  of 
hemotoxin  which  0.8  c.c.  possesses. 

b.  Examination  and  titration  0/4  luetic  sera  by  Citron's  method  (see  plate  II.) 

The  technical  details  of  the  test  are  as  follows: 

Three  est  tubes  are  assigned  for  each  test  and  placed  into  a  test  tube  rack.  The 
name  of  the  patient  is  written  upon  the  first  of  these  tubes.  Another  rack  contains  one 
tube  for  each  patient  and  labelled  accordingly.  In  addition  there  is  an  " antigen  tube," 
which  is  placed  into  the  first  rack  at  the  end  of  all  the  other  tubes;  also  a  "normal  extract," 
a  "system,"  "complement,"  and  "blood  control  tube,"  which  are  placed  into  the  second 
rack.  The  amount  of  syphilitic  antigen  required  for  the  entire  work  is  calculated  as 
follows.  For  each  test  0.3  of  antigen  is  required;  for  five  cases  (including  controls)  1.5 


Luetic 
extract. 

Serum. 

Comple- 
ment. 

Hemoly- 
sin;i  c.c. 

Sheep's 
blood. 

Result  of  hemolysis. 

I.    O.2 

o  .  2  Ser.  I.  Tabes  untreated; 

O.I 

i  :  1000 

i  c.c.  5% 

No  hemo-  ] 

without  luetic  history. 

lysis. 

2.    O.I 

o  .  i  As  above. 

O.I 

i  :  1000 

i  c.c.  5% 

No  hemo-  ) 

lysis. 

3.     ... 

0.2  *  As  above. 

O.I 

i  :  1000 

i  c.c.  5% 

Complete 

hemolysis.  J 

4.    0.2 

0.2  Ser  II.  Secondary  lues 

O.I 

i  :  1000 

i  c.c.  5% 

No  hemo- 

untreated. 

lysis. 

5.  o.i 

o  .  i  As  above. 

O.I 

i  :  1000 

i  c.c.  5% 

Incomplete      1 

hemolysis. 

6.    ... 

o  .  2  l  As  above. 

O.I 

i  :  1000      ice.  5% 

Complete 

hemolysis. 

164 


THE    TECHNIQUE    OF    COMPLEMENT    FIXATION. 


Luetic 
extract. 

<S£- 

Hemoly- 
sin;  i  c.c. 

Sheep's 
blood. 

Result  of  hemolysis. 

7.    0.2 

0.2  Ser.  Ill  Tabes.  Many         o.i           i  :  1000      i  c.c.  5% 

No  hemoly- 

inunction courses. 

sis. 

8.  o.i 

o  .  i  As  above.                                o  .  i 

i  :  1000 

i  c.c.  5% 

Complete 

hemolysis. 

r    +  + 

9.  ... 

o  .  2  As  above.                                o  .  i 

i  :  1000 

i  c.c.  5% 

Complete 

hemolysis. 

IO.    O.2 

0.2  Ser.  IV  Gallstones.                o.i       j   i  :  1000 

i  c.c.  5% 

Trace  of  bind- 

Lues seventeen  years  ago. 

ing;  almost  but 

Much   treatment.     No 

not  quite  com- 

symptoms for  ten  years. 

plete  hemolysi 

3 

II.    O.I 

o.i  As  above.                                o.i          i  :  1000      i  c.c.  5% 

Complete 

: 

hemolysis. 

12.     ... 

0.2*  As  above.                             o.i          i:  1000 

i  c.c.  5% 

Complete 

hemolysis. 

13.    0.2 

o  .  2  Negative  control  serum         o.i          i  :iooo 

i  c.c.  5% 

Complete 

(carcinoma  hepatis). 

hemolysis. 

14.     ... 

0.21  As  above.                              o.i          i  :  1000      i  c.c.  5% 

Complete 

hemolysis. 

15.  o.i 

o.i  Strongly  positive  con-         o.i          i  :  1000  '  i  c.c.  5% 

No  hemo-    ] 

trol  serum.     (Lues  malig- 

lysis. 

na). 

I 
( 

•f  +  +  + 

16.   ... 

o  .  2  As  above.                                o  .  i 

i  :  1000 

i  c.c.  5% 

Complete 

hemolysis.    j 

17.    O.2 

0.2   Weakly  positive  con-         o.i          i  :  1000      i  c.c.  5% 

Incomplete 

trol  serum.    Primary  lesion. 

hemolysis. 

18  

o.  2  *  Weakly  positive    con-       o.i           i  :  1000      i  c.c.  5% 

Complete 

trol  serum.    Primary  lesion. 

hemolysis. 

19.  0.4 

o  i 

i  :  looo      i  c.c.  5% 

Complete  hemolysis. 

Normal 

extract. 

20.    0.2 

o  .  2  Serum  I. 

O.I 

I        1000 

i  c.c.  5% 

Complete  hemolysis. 

21.    O.2 

0.2  Serum  II. 

O.I 

I       IOOO 

i  c.c.  5% 

Incomplete  hemolysis. 

22.    O.2 

o  .  2  Serum  III. 

O.  I 

I        IOOO 

i  c.c.  5% 

Complete  hemolysis. 

23.    O.2 

o  .  2  Serum  IV. 

O.  I 

I       IOOO 

i  c.c.  5% 

Complete  hemolysis. 

24.    0.2 

o  .  2  Negative  control  serum. 

0.  I 

I       IOOO 

i  c.c.  5% 

Complete  hemolysis. 

25.    O.2 

0.2  Strongly  positive  con- 

0. I 

I       IOOO 

i  c.c.  5% 

Complete  hemolysis. 

trol  serum. 

26.    O.2 

o  .  2  Weakly  positive  control 

O.I 

I        IOOO 

i  c.c.  5% 

Complete  hemolysis. 

serum. 

27      O   4. 

O.  I 

i  :  1000 

I  C.C.   q% 

Complete  hemolysis. 

28 

O    I 

I   I2OOO 

ICC     X>°7n 

Complete  hemolysis. 

O    I 

ICC     $°/r> 

No  hemolysis. 

icc   <;% 

No  hemolysis 

JU.       .    .    . 

SERUM    DIAGNOSIS    OF    SYPHILIS.  165 

c.c.  are  needed  +0.4  c.c.  for  the  antigen  control  tube  =  1.9  c.c.  in  all  or  2.0  c.c.  in  round 
numbers.  This  amount  is  diluted  with  normal  salt  solution  in  the  proportion  of  1 15  so 
that  8  c.c.  of  saline  are  added  (=2:10),  i  c.c.  of  this  dilution  contains  0.2  antigen  and 
1/2  c.c.  contains  o.i  antigen.  The  first  tube  (i,  4,  7,  10,  etc.,  in  diagram)  of  every  test 
therefore  receives  i  c.c.,  the  second  tube  1/2  c.c.,  the  third  tube  nothing,  the  antigen  tube 
(tube  19)  2  c.c.  Physiological  salt  solution  is  added  to  make  up  i  c.c.  in  each  tube; 
first  tube  nothing;  second  tube  1/2  c.c.;  third  tube  i  c.c.  of  saline. 

The  normal  extract  required  for  the  tubes  in  the  second  rack  is  similarly  estimated, 
0.2  c.c.  is  needed  for  each  test,  5  (tests) X 0.2=  1.0+0.4  for  the  antigen  control  tubes 
=  1.4  or  1.5  c.c.  in  round  numbers.  For  purposes  of  dilution  1 15,  6  c.c.  of  salt  solution 
are  added  and  i  c.c.  (  =  0.2  of  extract)  placed  into  each  of  the  tubes  (20  to  26)  and  2  c.c. 
into  the  normal  extract  control  test-tube  (tube  27).  In  this  series  also,  salt  solution  is 
added  to  make  up  equal  quantities  of  i  c.c.:  first  tube  nothing,  second  tube  1/2  c.c.,  third 
tube  i  c.c.;  antigen  tube  27,  nothing;  system  (28),  complement  (29)  and  blood  (30) 
control  tubes  each  i  c.c. 

The  second  ingredient  of  the  test  is  next  added,  i.e.,  the  respective  serum.  This  is 
not  diluted  but  added  directly;  0.2  c.c.  into  the  first  tube  of  each  test;  o.i  c.c.  into  the 
second;  0.2  c.c.  into  the  third  tube;  also  0.2  into  the  control  series  of  tubes  labelled  with 
the  patient's  names  in  the  second  rack.  Salt  solution  is  again  added  to  make  up  to  the 
equal  quantity  of  2  c.c.  in  each  tube,  thus:  0.8  c.c.  into  first,  o.  9  c.c.  into  the  second,  o.  8 
c.c.  into  the  third  tube,  and  0.8  c.c.  into  the  control  series;  nothing  into  the  antigen  tubes, 
i  c.c.  into  system,  complement,  and  blood  control  tubes. 

The  addition  of  complement  follows  next.  Each  tube,  except  the  blood  tube,  receives 
o.i  of  complement.  Thus  the  tubes  are  counted  and  if,  for  example,  ninteen  tubes  are 
present  19X0.1  c.c.  complement  is  taken,  or  in  round  numbers  2  c.c. 

Complement  is  always  diluted  i  :  10,  or  2  c.c.  complement  +  18  c.c.  saline,  so 
that  each  tube  except  the  blood  tube  (30)  receives  i  c.c.  of  this  diluted  complement 
Tube  (30)  receives  i  c.c.  of  saline  instead.  All  tubes  are  then  carefully  shaken  and 
the  racks  placed  into  the  incubator  for  one  hour. 

During  this  time,  the  hemolysin  and  washed  red  blood  cells  are  properly  diluted. 
The  red  blood  cells  are  made  up  in  a  5  per  cent,  suspension  of  which  each  tube  will 
receive  i  c.c.  Thus  in  the  present  test  thirty  tubes  exist,  requiring  30  c.c.  of  blood 
suspension;  since  i  c.c.  of  washed  blood  when  diluted  i:  20  will  supply  twenty  tubes, 
for  30  c.c.  about  i  1/2  c.c.  of  blood  will  be  required,  or  2  c.c.  will  make  40  c.c.  of  a 
5  per  cent,  blood  suspension. 

As  for  the  hemolysin,  its  titer  for  example  is  i  :  2000  and  it  is  employed  in  the  dilution 
of  i  :  1000.  Each  tube  except  the  blood  and  complement  tubes  will  receive  i  c.c. 
of  the  diluted  hemolysin;  i  c.c.  of  the  latter  if  diluted  properly  would  give  1000  c.c.; 
o .  i  of  the  hemolysin  which  is  the  smallest  amount  that  can  be  measured  out,  will  give 
100  c.c.  Every  tube  except  28  to  30  will  receive  i  c.c.  of  the  hemolysin  dilution  i  :  1000. 
Tubes  29  and  30  will  receive  none  (replaced  by  saline),  tube  28  will  receive  1/2  c.c.  of 
this  hemolysin  and  1/2  c.c.  of  saline. 

After  an  hour's  incubation,  each  tube  receives  i  c.c.  of  R.  B.  C.  and  i  c.c.  of  the 
hemolysin  just  mentioned.  If  it  is  desired  to  hasten  the  results,  it  is  advisable  to  mix  a 
sufficient  equal  quantity  of  R.  B.  C.  and  hemolysin  solution  (30  c.c.  of  each)  and  allow 
the  mixture  to  remain  in  the  incubator  for  a  short  time  before  the  hour's  incubation 

1  0.4  c.c.  of  luetic  serum  frequently  binds  complement  of  its  own  accord.  Experience  has 
shown  that  if  0.2  c.c.  does  not  bind  complement  and  0.2  c.c.  of  serum +  0.2  c.c.  of  antigen  does 
bind  complement,  the  unknown  serum  is  surely  of  luetic  origin. 


1 66  THE   TECHNIQUE    OF   COMPLEMENT   FIXATION. 

is  up.  Then  instead  of  adding  i  c.c.  of  these  ingredients  separately,  2  c.c.  of  the  mixture 
is  added  to  all  except  tubes  28  to  30.  Tubes  29  and  30  receive  i  c.c.  of  blood  and  i  c.c. 
of  saline  and  tube  28  i  c.c.  of  blood,  1/2  c.c.  of  hemolysin  and  1/2  c.c.  of  saline  which 
have  been  sensitized. 

The  various  strengths  of  the  resulting  reactions  are  differentiated  as 
follows : 

a.  Tubes  i   and   2   show  complete  absence  of   hemol-    i 

ysis:  +  +  +  +  {  Strongly 

b.  Tube  i  shows  complete  absence  of  hemolysis  and  positive. 

2  shows  faint  hemolysis :  +  +  + 

c.  Tube  i  shows  complete  absence  of  hemolysis  and 

2  shows  complete  hemolysis :  +  +  Weakly 

d.  Tube  i  shows  partial  hemolysis  and  f      positive. 

2  shows  complete  hemolysis:  + 

e.  Tube  i  shows   doubtful   binding   and  1    —     ,   r  , 

Doubtful. 
2  shows  complete  hemolysis :  ± 

/.   Tubes  i  and  2  show  complete  hemolysis:  —  ,  Negative. 

When  a  series  of  tests  is  to  be  performed,  it  is  advisable  to  include  in  the 
reaction  three  already  tested  sera,  one  strongly  positive,  another  weakly 
positive  and  a  third,  negative,  so  that  the  new  result  can  be  more  readily 
compared.  In  this  way  absolutely  reliable  and  constant  values  will  be 
obtained. 

Every  new  antigen  should  be  tested  for  four  weeks  before  its  practical  value  can  be 
assured.  During  this  month,  all  the  tests  should  be  done  with  both  the  old  and  new  ex- 
tract and  only  if  their  results  are  equal  should  the  new  extract  be  employed.  The  author 
is  in  the  habit  of  mixing  the  new  antigen  with  the  old  one  after  the  former  has  proved 
itself  efficient.  Occasionally  the  new  antigen  varies  in  strength  from  the  old  one.  In 
such  a  case,  if  stronger,  it  must  be  used  in  a  smaller  dose  (0.18  and  0.9)  or  if  weaker, 
must  be  used  in  larger  dose  (0.22  and  o.n).  Shaking  up  of  the  antigen  should  be 
strictly  guarded  against. 

In  order  to  control  the  effect  of  normal  liver  substances  contained  in  the  antigen,  an 
extract  is  prepared  in  an  analogous  method  from  normal  fetal  liver  (normal  antigen). 

A  strongly  positive  Wassermann  reaction  indicates  the  presence  of  a 
luetic  infection.  A  weakly  positive  result  can  be  similarly  interpreted  if  the 
serum  control  tube  (Tube  No.  3)  is  completely  hemolysed.  If,  however,  the 
latter  still  shows  some  non-hemolysed  red  blood  cells,  the  +  reaction  must 
be  considered  as  ±  or  a  reaction  of  indefinite  nature.  Only  exceptionally  are 
such  doubtful  reactions  found  in  perfectly  healthy  individuals,  although 
they  are  more  often  encountered  in  different  infectious  diseases  (typhoid, 
measles,  scarlet)  and  tumors.  A  positive  diagnosis  of  lues  should  never  be 
made  upon  a  ±  reaction.  On  the  other  hand  if  there  is  a  history  of  lues,  or 
clinical  evidences  of  the  same,  a±  reaction  is  to  be  interpreted  as  +  and 
should  warrant  further  specific  therapy.  As  an  end  result  of  specific  therapy 


SERUM    DIAGNOSIS    OF    SYPHILIS.  167 

a±  reaction  is  not  sufficient.     Not  before  an  absolutely  negative  reaction 
has  been  attained  should  specific  therapy  cease. 

Several  authorities  consider  only  such  tests  as  positive  where  there  is  complete  absence 
of  hemolysis.  This  principle  is  proven  as  incorrect  by  their  own  statistics;  a  great 
number  of  their  surely  syphilitic  cases  give  a  negative  reaction. 

If  the  third  tube  (serum  control)  does  not  hemolyse,  the  test  can  neither  be  considered 
as  positive  nor  negative.  Very  frequently  the  third  tube  of  very  strongly  positive  cases 
will  hemolyse  very  much  more  slowly  than  negative  cases;  these  tests  must  therefore 
remain  in  the  incubator  for  a  longer  period  than  the  negative  or  weakly  positive  ones  [ed]. 
and  until  the  serum  tube  is  completely  hemolysed. 

b.  Modifications  of  Wassermanrts  Technique. 

On  account  of  the  somewhat  complex  technique  of  the  reactions,  numer- 
ous attempts  have  been  made  to  simplify  the  test  in  one  way  or  another. 
The  greatest  difficulty  lay  in  the  preparation  of  a  suitable  antigen.  From 
the  sundry  modifications  and  improvements  made  in  this  respect,  perhaps 
the  most  important  was  announced  simultaneously  by  Landsteiner,  Muller 
and  Potzl,  and  Forges  and  Meier. 

They  showed  that  by  alcoholic  extraction  of  luetic  and  even  normal 
organs  of  human  beings  and  lower  animals,  substances  were  obtained 
which  could  be  used  as  a  substitute  for  the  aqueous  syphilitic  antigen.  The 
belief  therefore  arose  that  the  active  agents  in  the  luetic  extract  belong  to  the 
class  of  lipoids,  and  Forges  and  Meier  endeavored  to  isolate  them  from  the 
serum.  Thereupon  it  became  evident  that  lecithin  could  replace  the  antigen, 
but  only  up  to  a  certain  point.  Further  study  by  H.  Sachs  lead  to  the 
adoption  of  entire  formulae  for  artificial  antigens. 

The  new  principle  disclosed  by  these  discoveries  lead  to  many  modifications  in  the 
preparation  of  the  antigen,  the  main  advantage  of  which  consisted  in  bringing  the  reaction 
into  more  general  use  and  application.  The  previous  necessity  of  making  an  extract 
from  the  liver  of  a  luetic  fetus  somewhat  limited  this.  The  Wassermann  reaction  became 
in  a  short  period  of  time  much  more  popular,  although  one  could  not  adhere  to  it  with  the 
same  idea  of  specificity  as  before. 

Other  changes  in  the  reaction,  referred  to  the  serum  for  examination. 
H.  Sachs  demonstrated  that  the  inactivation  at  56°  C.  destroyed  a  great  part 
of  luetic  the  "reagine."  The  dispensation  of  the  latter  was  therefore  recom- 
mended. It  soon  became  evident,  however,  that  by  so  doing,  a  great  number 
of  normal  and  non-luetic  pathological  sera  gave  a  positive  reaction.  //  is 
best  therefore  that  this  modification  should  by  all  means  be  discarded. 

As  all  fresh  sera  contain  complement,  the  addition  of  guinea-pig's 
complement  seemed  superfluous  if  the  serum  for  examination  is  employed 
in  an  active  form.  The  following  combination  was  therefore  proposed: 

1.  Luetic  extract  or  one  of  its  substitutes. 

2.  Active  luetic  serum   (contains   luetic   "reagine"  +  complement).     One  hour  in 
incubator. 


1 68  THE    TECHNIQUE    OF    COMPLEMENT   FIXATION. 

3.  Inactive  hemolysin. 

4.  Red  blood  cells. 

In  view  of  the  above-mentioned  objections,  to  wit,  the  too  frequent  positive  results, 
this  modification  although  advised  by  divers  authorities,  Stern  and  others,  should  not 
be  employed. 

Not  only  the  addition  of  complement,  but  also  of  immune  hemolysin 
can  be  discarded,  because  every  serum  normally  contains  hemolytic  anti- 
bodies for  foreign  species  of  blood.  The  contraindication  for  the  trans- 
fusion of  foreign  blood  depends  upon  this  principle. 

Accordingly,  some  authors  advise  the  following  schemes: 
i.  Luetic  extract  or  its  substitute.  i.  Luetic  extract  or  its  substitute. 


2.  Inactive  luetic  serum  ("luesreagine"  + 
hemolysin). 


2.  Active  luetic  serum  (luesreagine  +  com- 
plement +  normal    hemolysin),     one 


3.  Complement.     One  hour  in  incubator.  hour  in  incubator. 

4.  Washed  erythrocytes  of  sheep.  j  3.  Washed  erythrocytes  of  sheep. 

The  advantage  of  these  modifications  is  supposed  to  exist  in  the  omission  of  the 
immune  hemolysin.  The  preparation  and  preservation  of  this  ingredient  is,  however, 
technically  so  simple  that  this  advantage  is  only  theoretical.  Bauer  believes  that  this 
change  is  preferable  to  the  classical  method  for  the  reason  that  with  the  latter,  the  x 
amount  of  normal  hemolysin  is  always  added  to  the  constant  amount  of  immune 
hemolysin,  thereby  resulting  in  a  different  quantity  of  the  same  in  each  test.  Experi- 
ence has,  however,  shown  that  the  faint  trace  of  normal  hemolysin  never  influences 
the  result  of  the  test.  At  times  so  little  normal  hemolysin  will  exist  in  a  patient's 
serum  that  it  becomes  necessary  to  add  some  serum  of  another  normal  patient.  Such 
manipulations  lead  to  new  difficulties  so  that  taken  all  in  all,  this  innovation  offers  no 
advantages  and  should  therefore  not  be  accepted. 

Brieger  and  Renz  have  recently  advised  the  substitution  of  potassium  chlorate  for  the 
immune  hemolysin.  Had  this  been  correct  the  biological  bases  of  the  Wassermann 
reaction  would  have  been  undermined.  Garbat  and  Munk  have,  however,  shown  that 
in  this  modification  KC1O3  is  entirely  inert  and  that  the  reaction  depends  upon  the 
normal  hemolysin  in  human  serum  against  sheep's  erythrocytes. 

Several  workers  in  this  field  believed  that  it  would  be  advantageous  to 
use  a  different  species  of  blood  in  place  of  sheep's  erythrocytes. 

The  only  suggestion  which  sounds  theoretically  correct  is  that  of  Noguchi,  who 
employs  human  erythrocytes  and  the  serum  of  a  rabbit  immunized  against  human 
red  blood  cells.  In  this  way  he  attempts  to  exclude  the  x  normal  hemolysins,  as  human 
serum  possesses  no  hemolysins  against  human  blood  cells. 

1.  Syphilis  extract  or  its  substitute.  i.  Syphilis  extract. 

2.  Inactive  syphilis-serum.         |  2.  Active    defibrinated    syphilitic    blood. 

3.  Complement   from  human   |  (Erythrocytes,    "reagine,"    comple- 

being    or  guinea-pig,     [    Syp  ment),  one  hour  in  incubator. 

.         .    .       .  serum.  ,.   . 

one  hour  in  incubator.       J  3.  Immune  hemolysin  of  rabbit  (injected 

4.  Immune  hemolysin  of  a  rabbit  against  j  with  human  blood). 

human  erythrocytes. 

5.  Washed  human  erythrocytes. 


SERUM    DIAGNOSIS    OF    SYPHILIS.  169 

From  a  practical  standpoint,  however,  no  distinct  advantage  is  offered  by  these 
modifications.  In  fact,  it  is  the  claim  of  Wassermann  and  his  pupils  that  by  the  use  of 
human  blood,  the  error  tends  towards  the  opposite  direction,  i.e.,  the  percentage  of  posi- 
tive results  obtained  are  higher  than  is  actually  the  case. 

The  number  of  modifications  have  become  so  numerous  that  almost 
every  one  employs  his  own  "method."  There  is  absolutely  no  necessity  for 
this,  as  an  innovation  justifies  its  existence  only  if  it  is  a  distinct  improve- 
ment, i.e.,  discloses  a  new  fact  or  radically  simplifies  the  old. 

It  is  the  classical  Wassermann  reaction  performed  in  the  original  manner 
which  has  taught  physicians  how  valuable  a  clinical  aid  it  is.  Their  knowl- 
edge has  not  advanced  a  step  further  even  with  all  the  new  changes.  A 
single  advantage  only  has  been  instituted  through  all  this  agitation,  and  that 
was  the  discovery  that  the  luetic  antigen  can  be  replaced  by  the  alcoholic 
extract  of  guinea-pig's  heart.  In  important  differential  diagnosis,  however,  even 
this  kind  of  extract  should  not  be  considered  as  specific  as  luetic  liver  antigen. 

For  general  work,  however,  its  employment  may  be  of  service. 

The  antigen  of  Landsteiner,  Muller  and  Potzl  is  prepared  as  follows: 

The  heart  of  a  guinea-pig  is  washed  free  of  blood,  its  muscular  part  finely  divided 
or  macerated  in  a  mortar  and  then  extracted  with  95  per  cent,  of  alcohol  for  several 
hours  at  60°  C.  One  gram  of  the  heart  substance  should  be  mixed  with  5  c.c.  of  the 
alcohol.  The  material  is  then  passed  through  filter-paper,  the  filtrate  being  kept  at 
room  temperature.  [The  editor  prepares  the  alcoholic  extract  by  simply  placing  the 
finely  divided  guinea-pigs'  hearts  into  95  per  cent,  alcohol  and  allowing  them  to  remain 
there  for  almost  four  weeks  for  purposes  of  extraction.  At  the  end  of  this  period  the 
alcoholic  solution  is  titrated  and  can  be  employed  as  antigen.] 

These  authors  also  employ  the  so-called  drop  method: 
Drop          Ten    drops    of   saline   and    i   drop    of  normal  guinea-pig's  serum  as 

Method.       complement  is  placed  into  each  test-tube.     The  individual  tubes  then 
receive  the  following  ingredients: 

First  tube:  One  drop  of  the  inactivated  serum  for  examination. 

Second  tube:  Same  as  one -I- 2  drops  of  the  alcoholic  heart  extract. 

Third  tube:  One  drop  of  inactivated,  surely  luetic  serum. 

Fourth  tube:  Same  as  three +  2  drops  of  alcoholic  heart  extract. 

Fifth  tube:  One  drop  of  inactive  normal  serum. 

Sixth  tube:  Same  as  five +  2  drops  of  alcoholic  heart  extract. 

Seventh  tube:  Two  drops  of  extract. 

The  tubes  are  well  shaken  and  placed  into  the  incubator  for  one  hour  at 
37°  C.  Then  i  drop  of  a  50  per  cent.  (!)  suspension  of  washed  sheep's 
erythrocytes  and  i  drop  of  hemolysin  (double  the  maximum  hemolytic 
titer)  are  added.  After  one-half  hour  in  the  incubator,  the  results  are  read 
off. 

Bauer  entirely  excludes  the  immune  hemolysin.     His  reaction  requires 
Bauer's        the  following  ingredients: 
Modification,   i.  Fresh  guinea-pig's  complement. 
2.  Alcoholic  organ  extract. 


1 70  THE   TECHNIQUE    OF    COMPLEMENT   FIXATION. 

3.  Five  per  cent,  sheep's  red  blood  corpuscles. 

4.  and  5.  The  inactivated  serum  for  examination  and  an  inactive  normal  control 
serum. 

Four  tubes  are  required  for  the  reaction: 

First  tube:  0.2  serum,  i.o  c.c.  organ  extract  in  dilution  i  :  5  and  i  c.c.  comple- 
ment i  :  10. 

Second  tube:  Same  as  i,  but  instead  of  organ  extract,  0.85  per  cent,  sodium  chloride. 

Third  tube:  Two-tenths  normal  serum,  organ  extract  and  complement  as  in  tube  i. 

Fourth  tube:  Same  as  third  tube,  but  instead  of  organ  extract  0.85  per  cent,  saline. 

The  tubes  are  placed  into  incubator  for  one-half  hour  and  then  i  c.c.  of  a  5  per  cent, 
red  blood  cell  emulsion  is  added. 

After  fifteen  to  forty-five  minutes  tubes  2,  3,  and  4  show  hemolysis,  while  tube  i 
shows  hemolysis  or  not,  depending  upon  the  absence  or  presence  of  syphilis. 

Lipemic  serum  is  not  suitable  for  the  reaction. 

Bauer  asserts  that  this  method  gives  results  identical  with  those  obtained  by  the 
Wassermann  tests.  Heinrichs,  Bering  and  others  confirm  Bauer's  findings. 

If  the  alcoholic  extract  made  from  luetic  or  normal  human  or  animal  organs  is  diluted 
with  physiological  saline,  a  milky  opalescent  solution  results.  The  grade  of  turbidity  of 
the  resulting  solution  depends  upon  the  rapidity  with  which  the  saline  for  dilution  is 
added.  If  the  first  15  to  20  drops  of  the  latter  are  added  slowly,  the  resulting  solution 
will  be  much  more  turbid  than  if  the  saline  is  added  quickly.  Sachs  first  observed 
this  phenomenon  and  stated  that  the  stronger  the  turbidity  the  more  active  is  the  power 
of  the  antigen  to  bind  complement. 

The  editor  has  worked  with  the  guinea-pig's  heart  extract  in  several 
thousand  tests  and  has  found  it  giving  perfect  results.  The  amount  usually 
used  is  0.2  to  o.i  c.c.  in  the  first  test-tube  and  o.i  to  0.05  in  the  second  test- 
tube  as  determined  by  titration.  When  the  antigen  is  diluted  (either  i :  5  or 
i :  10)  the  first  c.c.  of  saline  should  be  added  drop  by  drop  and  shaken,  thus 
producing  a  distinctly  opalescent  solution. 

The  author  refrains  from  describing  any  other  modifications  in  detail  as 
they  have  not  been  verified  sufficiently  to  merit  a  position  in  this  important 
field  of  serum  diagnosis.  This  holds  true  especially  for  the  recently  advised 
quick  and  easy  short  cuts  by  the  use  of  the  various  ingredients  dried  on 
paper.  In  order,  however,  that  one  may  acquaint  himself  with  these  modifica- 
tions, if  he  so  desires,  the  reference  of  their  original  publications  are  here 
given. 

Tschernogubow,  Berlin.  Klin.  Wochenschr.,  1908,  No.  47,  and  Deutsche 
Med.  Wochenschr.,  1909,  No.  15. 

Weidanz,  Deutsche  Med.  Wochenschr.,  1908,  No.  48,  Refer. 

Noguchi,  Journal  of  Americ.  Medic.  Associat,  1908,  No.  22,  u.  Munch. 
Med.  Woch.,  1909,  No.  10. 

Hecht,  Wien.  Klin.  Wochenschr.,  1908,  No.  50,  and  1909,  No.  10. 

Fleming,  Lancet,  1909,  4474. 

Stern,  Zeitschr.  f.  Immunitatsforschung,  1909,  Bd.  I. 

Bauer,  Deutsche  Med.  Wochenschr.,  1909,  No.  10. 


SERUM    DIAGNOSIS    OF    ECHINOCOCCUS    DISEASE. 

IV.  Serum  Diagnosis  of  Echinococcus  Disease. 


171 


The  technique  of  this  reaction  is  practically  the  same  as  described  for 
the  Wassermann  test. 

As  antigen  the  cystic  fluid  of  the  human  being  or  sheep  is  employed. 
The  latter  according  to  Weinberg  is  preferable,  as  human  hydatid  fluid 
sometimes  reacts  with  normal  serum. 

The  following  is  Weinberg's  outline  for  performing  the  test: 


Hydatid  fluid 
from  sheep. 

Inactive  serum 
from  patient. 

Complement. 

Hemolysin. 

Blood. 

Results. 

0.4 

0-5 

C.I 

2  X  hemoly  tic 

i  c.c.  5% 

o 

dose. 

sheep's  blood. 

0.4 

0.4 

O.I 

2  X  hemoly  tic 

i  c.c.  5% 

o 

dose. 

sheep's  blood. 

0.4 

o-3 

O.I 

2  X  hemoly  tic 

i  c.c.  5% 

Incomplete. 

dose. 

sheep's  blood. 

0.4 

0.2 

O.I 

2  X  hemoly  tic 

i  c.c.5% 

Complete. 

dose. 

sheep's  blood. 

0.4 

O.I 

2  X  hemoly  tic 

i  c.c.  5% 

Complete. 

dose. 

sheep's  blood. 

o  ^ 

O    I 

ICC     C% 

Complete 

dose. 

sheep's  blood. 

O    4. 

O    I 

2  X  hemoly  tic 

ICC     <C% 

Complete 

dose. 

sheep's  blood. 

O    3 

O    I 

2  X  hemolytic 

ICC     $°/n 

Complete. 

dose.  . 

sheep's  blood. 

O.2 

O.I 

2  X  hemoly  tic 

i  c.c.  5% 

Complete. 

dose. 

sheep's  blood. 

Bauer's  modification  as  employed  for  the  Wassermann  test  can  also  be 
employed  here. 

V.  The  Differentiation  of  Proteids  by  the  method  of  Neisser  and  Sachs. 

This  technique  varies  only  in  a  few  details  from  the  method  advanced 
later  on  by  Wassermann  and  Bruck  for  the  diagnosis  of  bacterial  infections. 

Neisser  and  Sachs  do  not  employ  a  constant  amount  of  complement  (o.i),  but  first 
titrate  the  complement  against  a  dose  of  hemolysin  double  its  hemolytic  titer.  For  the 
test  one  and  a  half  to  two  times  the  complement  titer  is  necessary.  The  hemolysin 
consists  of  the  serum  of  a  rabbit  immunized  against  ox's  blood.  This  hemolysin  acts 
both  for  ox's  and  sheep's  erythrocytes. 

The  amount  of  antiserum  (for  example  antihuman  serum)  used  for  the 
test,  is  influenced  by  two  factors. 

1.  An  excess  of  antiserum  can  interfere  with  the  fixation  of  complement. 

2.  The  antiserum  if  used  in  large  quantities  can  bind  complement  of  its 


172 


THE    TECHNIQUE    OF    COMPLEMENT   FIXATION. 


own  accord;  without  the  addition  of  the  human  serum,  for  example.  It  is 
therefore  best,  to  ascertain  by  titration,  the  smallest  quantities  of  antiserum 
which  may  satisfactorily  be  employed,  as  the  complement  fixation  test  must 
be  sufficiently  delicate  to  determine  o.oooi  c.c.  of  the  human  serum. 

Diminishing  amounts  of  antiserum  are  mixed  with  .0001  c.c.  of  human 
serum  and  o.i  c.c.  of  complement.  A  control  series  is  made  wherein  the 
human  serum  is  replaced  by  the  same  amounts  of  saline.  (The  quantity 
in  all  tubes  should  be  made  uniform  by  the  addition  of  normal  salt  solution, 
but  the  total  amount  of  fluid  in  each  tube  should  not  exceed  2.3  to  2.5  c.c.). 
The  tubes  are  incubated  for  i  hour  and  the  hemolytic  amboceptor  and  red 
blood  cells  added.  After  two  hours  at  37°  the  results  are  read  off.  The 
.0001  c.c.  of  the  serum  is  added  in  the  form  of  0.2  c.c.  of  a  i  :  2000  dilution. 

TABLE  III. 


Amounts  of  antiserum 
in  cubic-centimeters. 


Series     A     contains     antiserum  Series  B  (control)  contains  anti- 

+  o.oooi     c.c.     human     serum  serum +  0.2     c.c.     physiological 

(1:2000.02)4-0.1    guinea-pig's  saline  +  o.  i  of  guinea-pig's  serum 
serum. 


One  hour  at  37°. 
+  0.001  c.c.  of  amboceptor  +i 
c.c.  of  5  per  cent,  ox's-blood. 


One  hour  at  37°. 

+  0.001  c.c.  of  amboceptor  +  i  c.c. 
5  per  cent,  of  ox's-blood. 


Hemolysis. 

Hemolysis. 

O.  I 

Faint  trace. 

0.075 

Faint  trace. 

0.05 

0 

0-035 

0.025 

0 
0 

Complete. 

0.015 

Trace. 

O.OI 

Slight. 

0 

Complete. 

The  antiserum  itself  as  seen  in  the  control  series  (B)  does  not,  even  the 
amount  of  o.i  c.c.  (larger  quantities  never  come  into  consideration)  exhibit 
any  tendency  to  interfere  with  hemolysis.  On  the  other  hand,  series  (A) 
shows  that  the  larger  amounts  of  the  antiserum  do  not  bind  complement  as 
thoroughly  as  the  medium  doses.  The  zone  of  complete  complement  fixation 
lies  between  0.05  and  0.025  c.c.  of  the  antiserum.  It  is  advisable  as  a 
general  rule  to  choose  about  one  and  one-half  to  two  times  this  minimum 
quantity.  Thus  from  Table  III  it  can  be  noted  that  0.2  c.c.  of  a  i  :  6  dilution 
would  be  well  adopted  as  a  test  dose  for  complement  fixation.  If  it  is 
required  to  know  how  delicate  the  complement  fixation  reaction  can  be  with 
this  dose  of  antiserum,  the  following  experiment  (Table  IV)  is  undertaken : 

Diminishing  amounts  of  human  serum  are  mixed  with  a  constant  quan- 
tity of  complement  and  with  the  constant  test  dose  of  antiserum.  At  the 


SERUM    DIAGNOSIS    OF    ECHINOCOCCUS    DISEASE. 


173 


same  time  a  control  series  of  tubes  is  instituted,  in  which  the  antiserum  is 
substituted  by  salt  solution.  After  one  hour  of  incubation  at  37°  erythrocytes 
and  hemolysin  are  added.  Table  IV  illustrates  such  an  experiment. 


TABLE  IV. 


Amounts  of  human  serum 
in  cubic  centimeters. 

Series  A  contains  human  serum 
+  i  :  6X0.2  c.c.1      Antiserum, 
+  0.1  c.c.  of  guinea-pig's  serum. 

Series  B  (control)  contains  human 
serum  +  0.2     c.c.     physiological 
salt    solution  +  0.1    c.c.    guinea- 
pig's  serum. 

One  hour  at  37°. 

One  hour  at  37°. 

+  o.ooi      c.c.     of     amboceptor 
+  1  c.c.  of  5  per  cent,  ox's-blood. 

+  0.001   c.c.  of       amboceptor 
+  1  c.c.  of  5  per  cent,  ox's-blood. 

Hemolysis. 

Hemolysis. 

O.I 
O.OI 
O.OOI 
O.OOOI 
O.OOOOI 

o 

o 

0 
0 
0 

Slight. 
Complete. 

>  Complete. 

It  is  seen  from  the  above  table  that  o.ooooi  c.c.  of  human  serum  still 
suffices  to  give  a  partial  although  incomplete  fixation  of  the  complement. 
The  delicacy  of  the  antiserum  in  this  particular  instance  is  not  very  great. 
In  forensic  practice,  the  reaction  is  carried  out  as  shown  in  Table  IV,  but 
instead  of  the  human  serum,  the  solution  of  unknown  blood  stain  in  various 
dilutions  is  titrated.  Control  series  B  should  not  be  omitted,  because  here, 
any  foreign  substance  contained  in  the  extract  and  which  might  interfere 
with  the  reaction  can  be  detected. 


i  :  6X0.2  c.c.  means  0.2  c.c.  of  a  i :  6  dilution. 


CHAPTER  XV. 
PHAGOCYTOSIS.     OPSONINS  AND  BACTERIOTROPINS. 

i.  Phagocytosis. 

By  phagocytosis  is  meant  the  taking  up,  or  engulfing  of  foreign  substances 
by  certain  cells  (digestive  cells  or  phagocytes)  for  the  purposes  of  digestion. 
As  a  mode  of  nutrition,  this  is  well  known  to  exist,  normally,  in  the  lowest 
unicellular  animals  as  for  instance  the  amebae.  Intracellular  digestion  can, 
however,  be  traced  to  organisms  higher  in  the  scale  of  the  animal  kingdom, 
and  even  among  mammals  the  function  of  cell  ingestion  is  found,  although 
limited  in  a  sense,  to  a  definite  group  of  cells,  especially  those  derived  from 
the  mesoderm. 

The  inspiration  for  the  work  on  phagocytosis  and  the  greater  part  of  its 
theoretical  considerations  have  emanated  from  Metschnikoff  and  his  numer- 
ous pupils  at  the  Pasteur  Institute  at  Paris.  Metschnikoff  divides  the 
phagocytes  into  two  classes,  the  "sessile  or  fixed  phagocytes,"  and  the 
"wandering  phagocytes."  The  first  is  the  stationary  endothelial  lining  of 
blood  vessels,  and  lymph  spaces,  as  well  as  the  large  cells  of  the  spleen  pulp 
and  lymph  glands;  the  second,  consists  of  the  white  blood  cells  of  the  circula- 
tion. From  another  standpoint  the  phagocytes  are  divided  into  "micro- 
phages"  and  "macrophages."  The  former  are  practically  identical  with 
the  neutro-  and  eosinophile  polymorphonuclear  leucocytes,  while  the  latter 
present  no  distinct  group,  but  include  large  lymphocytes,  myelocytes,  giant 
cells,  etc.  The  cells  designated  as  sessile  phagocytes  also  belong  to  the 
class  of  macrophages.  The  size  of  the  cell  was  considered  by  Metschnikoff 
as  the  deciding  feature;  not  all  macrophages  are  mononuclear  as  generally 
believed.  Thus  for  example  macrophages  appearing  in  the  peritoneal 
fluid  of  guinea-pigs  frequently  possess,  like  the  giant  cells  of  the  tubercle, 
numerous  nuclei.  According  to  Metschnikoff  it  is  primarily  the  micro- 
phages  to  whom  the  function  of  bacterial  phagocytosis  is  allotted,  while  the 
macrophages  serve  for  the  purpose  of  ingesting  dead  or  moribund  tissue 
structure.  Still  there  are  certain  pathogenic  micro-organisms,  tubercle 
bacilli,  lepra  bacilli,  actinomyces,  which  are  favored  in  being  digested  by  the 
selective  macrophages.  The  evidence  of  phagocytosis  is  established  by 
mixing  either  in  vitro  or  vivo  the  substance  for  phagocytosis,  plus  the  phago- 
cytes, and  noting  the  changes  which  ensue;  [either  in  a  stained  or  unstained 
preparation].  The  phagocytes  of  the  guinea-pig's  peritoneal  cavity  are 


PHAGOCYTOSIS.  175 

especially  well  adapted  for  the  study  of  phagocytosis  in  vivo.     The  following 
experiment  of  MetschnikofT  may  serve  as  a  type. 

A  guinea-pig  receives  an  intraperitoneal  injection  of  goose's  blood.  Immediately 
following  this,  the  leucocytes  disappear  from  the  peritoneal  fluid.  This  is  due  partly  to 
a  destruction  of  leucocytes  (Phagolysis)  and  partly  because  the  leucocytes  are  repulsed 
and  settle  upon  the  peritoneal  wall.  In  one  to  two  hours  this  so-called  negative  phase 
is  overcome  and  there  is  an  increase  of  the  leucocytes,  especially  of  the  macrophages  in 
the  exudate  (Hyperleucocytosis).  Now,  the  leucocytes  can  be  seen  sending  forth  short 
protoplasmic  processes — pseudopodia,  by  means  of  which  the  erythrocytes  are  drawn 
into  the  phagocytes.  After  a  short  time  the  macrophages  are  filled  with  the  erythrocytes. 
At  first  the  ingested  cells  appear  normal ;  gradually,  however,  they  undergo  changes,  which 
are  clearly  visible  in  the  unstained  specimen,  indicative  of  a  disintegrating  process, 
within  the  body  of  the  phagocytes. 

The  same  phenomenon  as  described  for  goose's  erythrocytes  can  also  be 
observed  with  bacterial  bodies. 

In  order  to  exclude  the  possible  bactericidal  influences  of  the  serum,  it  is  advisable 
when  one  is  working  with  bacteria  which  are  readily  destroyed  as  cholera  vibrios,  to 
previously  induce  a  hyperleucocytosis  in  the  peritoneal  cavity.  The  guinea-pig  receives 
an  intraperitoneal  injection  of  10  to  20  c.c.  of  sterile  bouillon  or  aleuronatsolution.  In 
about  twelve  hours  hyperleucocytosis  takes  place,  and  a  capillary  pipette  inserted  into 
the  peritoneal  cavity  will  withdraw  a  thick  and  turbid  exudate. 

If  this  animal  is  injected  intraperitoneally  with  bacteria,  and  a  smear  of 
the  peritoneal  fluid  made  a  short  time  after  the  inoculation,  the  bacteria 
will  be  seen  lying  within  the  microphages.  This  important  fact  has  been 
variously  interpreted.  Pfeiffer  and  his  pupils  claim  that  the  bacteria  are 
first  destroyed  or  their  virulence  greatly  diminished  by  the  bactericidal 
power  of  the  serum  and  exudate,  and  "that  the  phagocytes  act  only  as  recepta- 
cles for  these  already  destroyed  bacteria.  Metschnikoff  believes  that  the 
phagocytes  take  up  the  living  bacteria  and  destroy  them,  thus  representing 
these  cells  as  the  most  important  weapons  of  the  organism  in  its  protection 
against  infection. 

"  Every  time  an  organism  that  has  lost  its  susceptibility  toward  a  partic- 
ular infective  agent,  either  on  account  of  a  natural  born  immunity  or  an 
artificially  attained  one,  comes  into  conflict  with  this  infective  agent,  a 
struggle  arises  between  the  latter  and  the  phagocytes  of  the  threatened 
individual.  It  is  the  phagocytes  that  appear  as  victors,  since  they  take  up 
the  bacteria  into  their  protoplasmic  bodies  and  digest  them,  thus  forever 
depriving  them  of  their  power  for  evil."  (Metschnikoff  cited  by  Levaditi). 

Critically  considered,  there  can  be  no  doubt  that  the  phagocytes  are  in 
principle  capable  of  dealing  with  living  virulent  bacteria.  At  the  same  time 
one  must  observe  that  the  opsonins  and  bacteriotropins  of  the  serum  soon 
to  be  discussed,  in  most  instances  previously  modify  the  living  bacteria  in 
some  way  at  present  still  unknown.  That,  however,  the  phagocytes  can 
ingest  bacteria  or  protozoa  which  are  alive  and  active,  has  been  demonstrated 


176  PHAGOCYTOSIS    OPSONINS  AND   BACTERIOTROPINS. 

by  Metschnikoff's  school.  Phagocytosis  experiments  were  undertaken 
with  motile  bacteria  and  spirilla.  On  microscopical  examination  it  was 
seen  that  a  phagocyte  was  in  the  act  of  taking  up  a  spirillum,  part  of  which 
was  engulfed  by  the  cell  while  the  remainder  was  still  outside  of  the  cell  and 
continuing  its  active  motility. 

Not  in  all  cases  does  phagocytosis  of  bacteria  lead  to  destruction  of  the  ingested 
microbes.  More  recently  different  experiments  seem  to  prove  that  simple  phagocytosis 
of  bacteria  must  not  be  considered  as  identical  with  death  of  the  same.  Furthermore, 
the  exudate  from  cases  of  anthrax  in  which  the  bacilli  lie  within  the  leucocytes,  can  still 
produce  fatal  anthrax  when  inoculated  into  animals. 

Vital  Staining  A  more  exact  understanding  of  the  bio-chemical  nature  of 
with  Neutral  phagocytic  digestion  has  been  offered  by  the  method  of  vital 
Red.         staining  with  neutral  red. 

Neutral  red  (used  as  a  i  per  cent,  solution  in  isotonic  saline)  is  a  chemical  dye  which 
stains  only  dead  cells  and  not  living  ones.  If  live  bacteria  and  phagocytes  are  brought 
into  contact  in  hanging  drop  preparations  (and  a  drop  of  the  stain  is  added  at  various 
intervals  to  a  different  mixture),  the  first  slide  shows  the  extracellular  living  bacteria 
unstained,  while  of  the  intracellular  bacteria,  a  part  remains  unstained  and  the  other 
colored  red. 

The  later  the  mixtures  are  stained,  the  more  numerous  are  the  intracellular  red  stained 
bacteria,  showing  that  the  injected  micro-organisms  remain  alive  for  a  short  time,  and  then 
die.  The  intracellular  bacteria  retain  their  stain  as  long  as  the  phagocytes  themselves 
remain  alive.  Later,  when  the  phagocytes  die,  the  formerly  red  bacteria  lose  their 
stain.  Metschnikoff's  explanation  of  the  red  staining  process  is  that  during  the  act  of 
digestion  by  the  phagocytes,  an  acid  ferment  is  liberated  which  gives  the  color  reaction 
with  the  neutral  red. 

For  many  years  Metschnikoff's  phagocytic  theory  opposed  the  conception 
of  Ehrlich  and  also  Pfeiffer  in  relation  to  the  importance  of  amboceptor  and 
complement  in  the  mechanism  of  immunity.  It  would  be  out  of  place  here 
to  review  the  various  experiments  performed  and  offered  on  each  side  in 
explanation  of  its  standpoint.  Suffice  it  to  say  that  Metschnikoff  denied  the 
existence  of  free  complement  within  the  animal  organism.  He  moreover 
claimed  that  the  complement  was  found  normally  only  in  the  phagocytes  and 
hence  called  it  "cytase,"  differentiating  the  two  phagocyte  groups  as  "micro- 
and  macrocytase."  The  "cytase"  is  liberated  when  the  phagocytes  are 
broken  up.  The  amboceptors  are  considered  as  split  products  of  the 
phagocytes  and  known  by  Metschnikoff  as  "fixators." 

2.  Opsonins. 

In  recent  years  the  closer  relationship  which  has  arisen  between  the  fol- 
lowers of  phagocytic  and  humoral  theories  was  made  possible  by  the  fact  that 
Denys  and  Leclef,  Leishmann,  Wright  and  Douglas  and  others,  demonstrated 
that  phagocytosis  occurs  in  most  cases  only  in  the  presence  of  serum.  If  the 
phagocytes  are  thoroughly  washed,  so  that  they  are  entirely  serum-free, 


OPSONINS. 


177 


phagocytosis  will  not  take  place,  or  will  do  so  imperfectly.  The  belief  of 
some  authors  that  "spontaneous  phagocytosis"  without  serum  was  alto- 
gether impossible,  was  disproved,  especially  by  Lohlein.  The  manner  in 
which  the  serum  acts,  whether  it  stimulates  the  digestive  activity  of  the 
leucocytes  or  whether  it  so  changes  the  bacteria  that  they  can  more  readily 
be  taken  up  by  the  phagocytes,  has  been  settled  in  favor  of  the  latter  view 
through  researches,  especially  of  Wright  and  his  followers  as  well  as  by 
Neufeld.  The  substances  within  the  serum  which  thus  modify  the  bacteria 
have  been  designated  by  Wright  as  "opsonins."  ("opsono"  =  I  prepare 
food  for). 

Opsonins  are  demonstrated  by  mixing  bacteria,  serum  and  washed 
leucocytes,  allowing  this  mixture  to  remain  in  the  incubator  for  a  short  time, 
and  then  staining  smear  preparations.     Wright  then  counts  a  certain  num- 
ber of  leucocytes  and  the  number  of  bacteria  found  within  these  leucocytes. 
The  relation  between  this  number  of  ingested  bacteria  and  the 
The  Opsonic  counted  number  of  phagocytes  is  designated  as  the  phagocytic 
Index.       count.     Wright    compared    the    phagocytic    counts    of  infected 
individuals  with  those  of  normal  persons  and  found  that  those  of 
the  former  were  much  lower.     The  relation  existant  between  the  two  he  expressed 
in  the  form  of  a  fraction  and  that  is  known  as  the  opsonic  index.     Thus  a  smear 
made  from  a  mixture  of  equal  parts  of  an  emulsion  of  staphylococci,  leuco- 
cytes and  the  patient's  serum  showed  for  example  75  cocci  to  100  leucocytes; 
while  one  made  from  a  mixture  of  equal  parts  of  the  same  bacterial  emulsion, 
leucocytes,  but  a  normal  individual's  serum  demonstrated  150  bacteria  to  100 
leucocytes.     The  opsonic  index  of  the  patient's  serum  would  therefore  be 
one-half  (0.5). 

According  to  Wright,  the  opsonic  index  expresses  the  animal's  resistance 
against  infection.  He  believes  that  a  low  opsonic  index  for  a  given  bacter- 
ium indicates  a  susceptibility  on  the  part  of  the  individual  for  that  particular 
infective  agent.  Furthermore,  the  opsonic  index  he  claims  can  be  used  as  an 
aid  in  the  diagnosis  of  infectious  diseases,  inasmuch  as  opsonins  are  specific. 
Thus  the  opsonic  index  in  a  tuberculous  individual  is  low  only  for  the  tuber- 
cle bacillus  and  not  for  other  bacteria. 

When  an  animal  is  immunized,  its  opsonic  index  toward  the  respective 
bacterium  is  considerably  increased.  The  question  has  been  asked  whether 
the  immune  opsonins  formed  during  this  process  are  identical  with  the  nor- 
mal opsonins.  Wright  and  a  number  of  the  more  recent  authorities  believe 
that  they  are  different.  Neufeld,  who  discovered  these  immune  opsonins 
independently  of  Wright,  named  them  Bacteriotropins,  and  pointed  out 
that  while  the  normal  opsonins  are  destroyed  when  heated  to  56°,  the  bacterio- 
tropins  remain  unharmed.  As  yet  the  exact  nature  of  the  immune  as  well  as 
of  the  normal  opsonins  has  not  been  clearly  defined.  It  is  still  a  matter  for 
investigation  whether  in  the  case  of  opsonins  one  is  dealing  with  entirely  new 

12 


PHAGOCYTOSIS    OPSONINS  AND    BACTERIOTROPINS. 


substances  or  whether  they  are  the  old  well-known  bodies  like  the  agglutinins, 
complements  and  amboceptors  with  a  new  action. 

The  fact  that  the  opsonic  index  is  raised  by  immunization  while  it  is 

usually  found  diminished  during  spontaneous  infection  in  man,  lead  Wright 

to  believe  that  good  results  may  be  obtained  by  increasing  the 

Increase  of    opsonic  index  of  the  already  infected  individual  by  means  of 

Opsonic  Index  immunization.     In  this  way  he  thought  the  patient's  pre- 

by  Immuni-   disposition  to  the  particular  infection  would  be  overcome,  with 

the  consequent  obtention  of  the  essential  requirements  for  a 

cure.     Wright's  experiments  showed  that  the  opsonic  index 

could  be  increased  by  injection  of  extremely  small  doses  of  dead  bacteria 

(Wright's  vaccines.) 

If  an  individual  suffering  from  an  acne  or  furunculosis,  and  who  has  a  low  opsonic 
index  for  the  staphylococcus,  is  injected  with  a  very  small  number  of  staphylococci,  his 
opsonic  index  sinks  still  more  for  a  short  period  after  the  inoculation  (negative  phase). 

This  is  explained  by  the  fact  that 
the  injected  bacteria  absorb  the 
existing  opsonins.  New  opsonins 
are  however  then  produced,  which 
immediately  make  up  for  the  loss 
occasioned  during  the  negative 
phase,  with  the  result  that  after 
several  days  there  is  an  increase  of 
the  opsonic  index  (positive  phase) 
which  lasts  for  a  short  time.  Then 
the  index  again  begins  to  fall,  as 
the  stimulus  for  the  formation  of 
opsonins  is  transitory.  It  usually 
sinks  to  below  the  normal  level, 


Opsonic 
Index  ]5 

!.» 

13 
12 
1.1 

Normal  1-° 

A 

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7 

V 

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0.5 

9 

10 

.11 

12 

13 

IH 

15 

16 

17 

18 

19 

20 

21 

22 

CHART  5. — Curve  of  the  opsonic  index  following  the 
inoculation  of  a  small  dose  of  staphylococcus  vaccine. 
The  arrow  indicates  the  time  of  injection. 

only  to  rise  again  to  a  point  slightly 

above  the  normal,  where  it  remains  stationary.  This  irregular  curve  represents  the 
typical  course  of  the  opsonic  content  of  the  blood  after  a  vaccine  injection;  apart  from 
this  characteristic  picture  numerous  exceptions  exist.  Thus  by  the  use  of  very  minute 
bacterial  doses,  the  negative  phase  immediately  following  the  injection  is  entirely 
absent.  Reversely  very  large  doses  exhibit  a  prolonged  negative  phase. 

Wright  graphically  represents  these  variations  in  the  opsonic  index  by 
charts,  an  example  of  which  is  given  here  (Chart  5) . 

In  order  that  the  therapeutic  effect  may  persist,  it  is  advisable  to  repeat 
the  inoculation.  A  new  injection  should  be  given  at  the  height  of,  or  during 
the  positive  phase,  as  an  inoculation  repeated  during  a  negative  phase  will 
result  in  further  depression  of  the  index  unto  a  very  low  level.  It  is  even 
possible  in  this  way  to  harm  the  patient.  The  poor  results  obtained  during 
the  first  era  of  tuberculin  treatment  can,  according  to  Wright,  be  attributed 
to  the  failure  of  this  observation.  It  is  the  production  of  cumulative  positive 
phases  that  is  the  aim  of  vaccine  treatment.  (Chart  6). 


OPSONINS. 


179 


Wright  and  his '  co-workers  have  noticed  that  an  increase  in  the  opsonic 
index  usually  runs  parallel  with  an  improvement  in  the  condition  of  the 
patient. 

Inasmuch  as  an  increase  in  the  opsonic  index  is  occasioned  by  introduc- 
ing into  the  general  system  even  a  very  small  number  of  bacteria,  it  seems 
probable  that  such  spontaneous  inoculation  will  take  place  during  the  course 
of  an  infectious  disease.  In  fact,  a  spontaneous  rise  in  the  opsonic  index  is 
observed  during  convalescence  or  after  the  crisis  of  an  infection.  A  high 
index  is,  however,  also  noticed  at  other  times,  for  example  tuberculous  individ- 
uals show  a  higher  index  than  normal  persons.  Wright  explains  this  by  the 


30 
29 
28 
27 
26 
25 
24 
23 
22 
21 
20 
19 
18 
17 
16 
15 
14 
13 
12 
11 
10 
9 
8 
7 
6 
5 
4 

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CHART.  6. — Opsonic  curve  during  treatment  with  New  Tuberculin. 

so-called  "auto-inoculation;"  for  example  after  moderate  exercise,  or  work, 
tuberculin  is  liberated  from  the  tuberculous  focus  and  in  this  way  acts  like  a 
therapeutic  injection  of  tuberculin,  i.e.,  the  index  will  be  raised.  Therefore, 
an  excessively  high  opsonic  index  is  of  just  as  great  diagnostic  value  as  a  low 
one.  Wright  furthermore  believes  that  constant  irregularities  or  variations 
in  the  height  of  the  opsonic  curve  serve  as  plausible  evidence  for  the  exist- 
ence of  infection,  because  under  normal  circumstances  the  curve  should 
remain  at  a  level.  Not  infrequently,  however,  cases  come  under  observation 
where  in  spite  of  a  distinct  evidence  of  the  existence  of  an  infection  the  opsonic 
index  remains  normal.  In  such  instances  for  some  reason,  the  bacteria  and 
their  products  do  not  reach  the  general  circulation  and  therefore  no  occasion 
is  offered  for  either  an  elevation  or  sinking  of  the  opsonic  index.  Wright  and 
Freeman  were  able  to  show  that  all  active  and  passive  motions  of  an  infected 
joint,  as  well  as  any  vascular  changes  which  induce  a  flow  of  lymph  toward 


i8o 


PHAGOCYTOSIS    OPSONINS  AND    BACTERIOTROPINS. 


Dpsanic 
Index. 

2.6 
2,4 
2.2 
2.0 
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1,6 
14 
7,2 
Normal    1  0 
0,B 
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Gono-0'psonic   Index.      •    •     • 

Tuberculo-Opsonic  Index      -o—  o—  o 

1 

1     , 

1 

CHART.  7. — Increase  in  the  opsonic  index  for  the  gonococcus  brought  about  by 
Bier's  Hyperemia. 


Feb. 


CHART.  8. — Tuberculin  auto-inoculation  following  physical  examination  and  massage. 
(Tuberculous  Lymph-adenitis.) 


TECHNIQUE    FOR    DETERMINATION    OF    OPSONIC    INDEX.  l8l 

the  focus  of  infection,  lead  to  auto-inoculations,  which  are  manifested  in  a 
change  of  the  opsonic  index.  Such  artificial  production  of  auto-inoculation 
can  be  employed  in  various  forms  as  a  means  of  diagnosis :  thus,  in  articular 
rheumatism,  massage;  in  pulmonary  tuberculosis,  breathing  exercises;  in 
laryngeal  diseases,  loud  reading;  and  in  tuberculosis  of  the  lower  extremities, 
active  gymnastics  will  occasion  changes  in  the  opsonic  curve. 

An  example  is  given  in  Chart  7.  The  patient  was  a  woman  with  a  swollen  wrist  joint. 
In  order  to  decide  whether  this  was  a  gonorrheal  or  tuberculous  process,  the  opsonic 
index  was  taken  and  found  to  be  0.94  to  0.97  for  the  gonococcus  and  1.03  to  1.35  for  the 
tubercle  bacillus.  As  these  figures  differed  very  slightly  from  the  normal,  the  test  was 
repeated,  but  this  time  after  Bier's  hyperemia  had  been  applied  and  the  forearm  placed 
into  warm  water  for  one  hour.  The  opsonic  index  for  the  tubercle  bacillus  remained 
the  same,  while  that  for  the  gonococcus  had  undergone  marked  variations. 

A  similar  experiment  with  a  woman  having  tuberculous  lymphadenitis  is  given  in 
Chart  8. 

Wright  makes  use  of  these  variations  of  index  caused  by  auto-inoculation 
in  determining  the  prognosis  of  a  case.  An  infection  is  only  then  considered 
cured  when  artificial  auto-inoculation  is  no  longer  -possible. 

The  Technique  for  the  Determination  of  the  Opsonic  Index. 

For  the  determination  of  the  opsonic  index  are  required, 

1.  Serum  of  the  patient. 

2.  Serum  of  the  normal  individual  (as  control). 

3.  Washed  blood  cells  (Leucocytes). 

4.  Bacterial  emulsion. 

The  blood  serum  is  obtained  from  the  finger  tip  at  the  root  of  the  nail. 
It  is  most  efficacious  to  first  produce  a  hyperemia  of  this  part  by  constricting 


FIG.  18.  FIG.  19.  FIG.  20. 


the  finger  either  with  a  narrow  gauze  bandage  or  a  small  soft  rubber  tube 
(editor  has  found  the  latter  much  more  convenient).  The.  prick  is  then 
made  with  a  needle  or  finely  drawn  out  glass  tube.  The  blood  flows  sponta- 
neously and  is  collected  into  one  of  Wright's  capillary  tubes  (Fig.  19)  approxi- 


182 


PHAGOCYTOSIS    OPSONINS   AND    BACTERIOTROPINS. 


mating  the  curved  end  of  the  latter  to  the  blood  (Fig.iS).  The  straight 
capillary  end  of  the  tube  (away  from  the  blood)  is  then  gently  warmed  in  a 
small  flame  and  sealed.  The  tube  is  laid  down  flat,  and  allowed  to  cool;  in 
so  doing  the  blood  is  sucked  back  from  the  unsealed  capillary  end;  next 
this  end  may  also  be  sealed  in  the  tip  of  the  flame.  The  blood  then  coagu- 
lates and  the  serum  separates  off.  The  separation  of  the  latter  may  be 
hastened  by  centrifugalization  for  a  short  time. 

In  order  to  obtain  leucocytes,  a  small  test  tube  which  holds  3  to  4  c.c.  is 
filled  2/3  with  a  1.5  per  cent,  solution  of  sodium  citrate,  and  about  6  to  7 
drops  of  blood  from  a  healthy  individual  are  collected  into  this  solution 
(Fig.  21).  The  tube  is  inverted  several  times  to  thoroughly  mix  the  blood  so 


FIG.  21. 


FIG.  22. 


that  the  citrate  by  precipitating  the  calcium  salts  of  the  blood,  effectively 
prevents  coagulation.  The  suspension  is  centrifugalized  until  the  corpuscles 
are  thrown  down  and  a  distinct  white  layer  (leucocytes)  is  seen  upon  the 
surface  of  the  red  cells  (Fig.  22).  The  clear  supernant  citrate  solution  is 
pipetted  off,  care  being  taken  not  to  disturb  the  white  layer.  Some  of  the 
0.85  per  cent,  saline  is  added,  mixed  and  again  centrifugalized.  The  wash- 
ing with  normal  saline  solution  is  repeated  once  or  twice  and  as  much  of  the 
clear  liquid  as  possible  is  finally  removed;  the  remaining  cells  are  thoroughly 
mixed  and  in  this  form  are  ready  for  use. 

The  bacterial  emulsions  with  the  exception  of  the  tubercle  bacillus  are 
made  from  agar  cultures;  the  growths  of  gram  +  cocci  may  be  as  old  as 
twenty-four  hours,  while  the  coliform  organisms  and  the  gram  — cocci 
are  preferable,  if  only  four  to  ten  hours  old,  the  younger  the  better. 
A  loopful  of  culture  from  an  agar  tube  is  thoroughly  rubbed  up  with  several 
drops  of  salt  solution  in  a  watch-glass  by  means  of  a  small  glass  pestle.  The 
salt  solution  is  best  added  very  gradually,  drop  by  drop,  thus  making  a  more 
perfect  emulsion.  This  may  then  be  advantageously  centrifugalized  for  a 
varying  period,  to  bring  down  the  large  clumps.  The  supernatant  opalescent 


TECHNIQUE    FOR    DETERMINATION    OF    OPSONIC    INDEX. 


portion  is  taken  off  for  use,  thoroughly  mixed,  and  if  necessary  diluted. 
Emulsions  of  coliform  organisms  are  more  easily  made.  Frequently  it  is 
sufficient  to  rub  up  with  the  platinum  loop  a  loopful  of  such  bacteria  on  the 
side  of  a  small  test  tube  containing  saline.  The  proper  thickness  of  the 
resulting  emulsion  varies.  As  a  rule,  bacillary  emulsions  are  required  to 
have  a  thicker  appearance  to  the  naked  eye  than  coccal 
ones.  The  latter  should  be  only  slightly  opalescent. 

In  order  to  make  a  satisfactory  tubercle  emulsion, 
a  more  elaborate  method  is  necessary.  The  dead  and 
dried  tubercle  bacilli  are  employed  for  this  purpose.  A 
portion  of  these  bacilli  is  very  thoroughly  triturated  in 
an  agate  mortar,  or  between  two  slides,  or  in  a  grinder 
devised  for  this  purpose,  at  first  alone  and  then  with  1.5 
per  cent,  salt  solution  added  drop  by  drop.  In  this  way 
a  paste,  and  subsequently  a  comparatively  thick  emul- 


Leucocytes 
Air  bubble 
Bacilli 

Mark    •£     Air  bubble 
Serum 


FIG.  23, 


FIG.  24. 


sion  is  made.     For  use,  a  small  portion  of  the  resultant  emulsion  is  centri- 
fugalized  until  only  the  upper  layers  are  fairly  opalescent. 

These  upper  layers  are  pipetted  off,  and  thoroughly  mixed.  A  smear  of 
this  should  be  made  and  stained  in  order  to  observe  that  the  emulsion  is  free 
from  clumps  and  not  too  thick.  Such  an  emulsion  sealed  up  in  a  glass  tube 
and  sterilized  at  60°  C.  for  i  hour  can  be  kept  for  about  one  week. 


184  PHAGOCYTOSIS    OPSONINS  AND    BACTERIOTROPINS. 

Streptococci  may  similarly  be  rubbed  up  in  a  mortar  with  0.85  per 
cent,  salt  solution  and  then  centrifugalized.  As  a  rule,  however,  vigorous 
pipetting  into  a  watch  glass  with  subsequent  centrifugalization  for  a  few 
minutes  is  sufficient  to  remove  the  chains  and  leave  a  satisfactory  emulsion. 

If  several  specimens  of  blood  are  to  be  examined  it  is  best  to  put  up  a 
"trial  trip"  and  do  a  preliminary  phagocytic  count  in  order  to  test  the 
strength  and  condition  as  regards  clumping  of  the  emulsion.  The  phagocy- 
tic count  should  be  for  tubercle  between  1.5  to  2  per  cell  and  for  other  organ- 
isms not  less  than  3  per  cell.  Accordingly,  further  dilution  or  concentration 
of  the  emulsion  is  necessitated.  The  pipettes  employed  for  the  opsonic 
index  should  be  about  16  cm.  long  and  made  from  glass  tubing  about  5/16 
of  an  inch  in  diameter.  They  should  all  be  approximately  of  the  same 
caliber  and  but  slightly  tapering  toward  the  point.  The  piece  of  tubing 


FIG.  25. 

should  tightly  fit  the  rubber  nipple  or  bulb  available.  For  use,  the  capillary 
end  should  be  cut  square  and  the  pipettes  marked  with  a  paraffin  pencil 
about  3/4  of  an  inch  from  their  extremity.  The  content  as  far  as  this 
mark  is  the  unit  of  volume  in  each  case. 

The  rubber  nipple  is  now  held  between  thumb  and  forefinger  and  gently 
compressed,  the  capillary  end  introduced  into  the  well  mixed  blood  cells 
and  the  unit  volume  drawn  up  by  slightly  relaxing  the  pressure  on  the  bulb. 
Next  a  tiny  bubble  is  allowed  to  enter,  then  an  equal  volume  of  the  emulsion, 
followed  by  another  tiny  bubble  which  latter  is  succeeded  by  an  equal 
volume  of  serum.  By  gentle  pressure  on  the  bulb  the  several  volumes  are 
ejected  upon  a  clean  glass  slide,  and  thoroughly  mixed  by  alternately  sucking 
the  mixture  into  the  pipette  and  squeezing  it  out  again  upon  the  slide.  It  is 
enough  to  repeat  this  action  three  times.  Then  the  mixture  is  drawn  up 
into  the  pipette,  the  end  sealed  in  a  small  pilot  flame,  the  pipette  placed  into 


TECHNIQUE    FOR    DETERMINATION    OF    OPSONIC    INDEX.  185 

the  opsonizer  (Fig.  24)  and  the  time  noted.  This  operation  is  repeated 
with  each  serum. 

Coliform  organisms  and  the  gram-cocci  should  be  incubated  not  longer 
than  six  to  eight  minutes.  Tubercle  bacilli  and  other  organisms  require 
fifteen  minutes  more  or  less,  according  to  the  strength  of  the  emulsion. 

The  pipettes  are  then  withdrawn  in  the  same  order  in  which  they  were 
placed  into  the  opsonizer.  The  contents  of  each  are  blown  out  on  to  a  slide 
and  very  carefully  mixed  as  before  (Fig.  25).  The  entire  quantity  is  divided 
between  two  or  three  slides  and  several  smears  are  made,  the  best  one  being 
selected  for  counting.  These  slides  should  previously  have  been  rough- 
ened with  very  fine  (oo)  emery  paper,  cleaned  with  a  duster,  and  should  rest 
on  their  concave  surface  so  that  the  smear  is  made  on  the  convex  side.  (It 
will  be  noticed  that  a  slide  can  be  made  to  rotate  if  resting  on  one  surface 
(convex),  but  does  not  do  so  when  resting  on  the  concave  surface).  The 
smears  are  best  made  by  means  of  the  edge  of  a  broken  slide  with  a  slightly 
concave  edge.  This  "spreader"  (Fig.  26)  is  made  by  sharply  breaking  a 


FIG.  26. 

glass  slide  at  about  its  middle,  this  being  facilitated  by  scratching  the  edges 
of  the  slide  with  a  glass  cutter  at  the  point  where  it  is  desired  to  break  it. 
The  editor  has  broken  as  many  as  twenty  to  thirty  slides  before  a  proper 
spreader  was  obtained.  It  pays  to  do  this,  because  upon  the  sharpness  of 
the  fracture  and  cleanliness  of  the  spreader  depends  the  edge  of  the  film,  and 
secondarily  the  ease,  rapidity,  and  accuracy  of  the  count.  If  the  film  be 
well  made,  it  will  have  a  straight  edge  within  which  will  be  found  practically 
all  the  leucocytes,  as  they  are  larger  than  the  red  blood  cells,  and  therefore 
dragged  to  the  end  of  the  film. 

The  preparations  are  fixed  in  a  saturated  solution  of  corrosive  sublimate 
for  two  or  three  minutes,  washed  with  water,  and  stained  with  methylene 
blue  or  carbol-thionin  (1/4  per  cent,  thionin,  and  i  per  cent,  carbolic  acid). 
Carbol  thionin  is  by  all  means  preferable.  It  should  be  slightly  diluted  and 
warmed  before  being  poured  upon  the  slide.  Here  it  is  allowed  to  remain 
for  several  minutes,  then  washed  off  in  water,  and  the  slide  dried  with  filter- 
paper.  The  tubercle  films  are  best  fixed  with  formalin  vapor,  stained 
with  hot  carbol  or  aniline  fuchsine,  decolorized  in  2.5  per  cent,  of  H2SO4, 


i86 


PHAGOCYTOSIS    OPSONINS  AND    BACTERIOTROPINS. 


treated  with  4  per  cent,  acetic  acid  to  dissolve  the  erythrocytes  and  counter- 
stained  with  1/2  per  cent,  of  methylene  blue  in  1/2  per  cent,  of  sodium  car- 
bonate. It  is  most  important  that  tubercle  films  be  carefully  stained 
because  it  is  desirable  to  color  every  bacillus  and  yet  not  break  up  the 
leucocytes  (Fig.  27). 

With  a  i/ 1 2  inch  oil  immersion  lens  a  minimum  number  of  one  hundred 
polymorphonuclear  leucocytes  are  now  examined  and  the  number  of 
microbes  they  contain  enumerated. 


FIG.  27. — Phagocytosis  of  tubercle  bacilli. 

Similar  calculation  is  undertaken  with  the  normal  control  serum.  The 
fraction  obtained  by  dividing  the  number  of  bacteria  contained  in  100  cells, 
on  the  patient's  slide,  by  the  number  in  100  cells,  on  the  normal  slide,  gives 
the  opsonic  index  of  the  patient's  serum. 

For  example,  the  normal  individual  has  284  and  the  patient  262  bacteria 
in  100  cells,  the  fraction  which  gives  the  patient's  opsonic  index  would  be 
262/284  or  0.92. 

The  principle  of  Wright's  technique  is  simple,  but  it  requires  a  great 
deal  of  practice  before  it  is  mastered.  Only  then  are  the  results  reliable. 
One  must  remember  the  same  principles  when  counting  the  control  slide  as 
when  the  patient's  film  is  counted.  If  in  the  last  case,  for  instance,  the  cocci 
situated  on  the  edges  of  the  cells  are  not  included  in  the  count,  the  same 
should  also  be  excluded  in  the  first  case.  The  absolute  count  is  of  no 
importance.  It  is  the  relative  proportion  which  is  significant.  • 

As  a  normal  control,  it  is  best  to  take  the  average  of  the  phagocytic 


WRIGHT'S  VACCINE  TREATMENT.  187 

counts  of  a  series  (3  to  4)  of  normal  sera  or  first  equally  mix  the  different 
sera,  and  take  the  phagocytic  count  of  the  pool. 

Normal  sera  should  not  differ  from  one  another  in  a  tubercular  opsonic 
estimation  by  more  than  10  per  cent. 

Wright's  Vaccine  Treatment. 

As  has  been  said,  the  principle  of  Wright's  vaccine  treatment  depends 
upon  the  immunization  with  small  doses  of  dead  bacteria,  so-called  vaccines, 
whereby  the  opsonic  index  of  the  individual  is  raised.  This  is  usually 
associated  clinically,  with  improvement  in  the  patient's  condition. 

The  effect  of  the  immunization  according  to  Wright  depends  upon: 

1.  Individual  reaction  of  the  patient. 

2.  Preparation  of  the  vaccine. 

3.  Dosage  and  form  of  application. 

The  individual  reaction  of  the  patient  can  be  measured  by  the  opsonic 
index. 

As  for  the  preparation  of  the  vaccine,  Pasteur's  contention  that  a  vaccine 
must  necessarily  be  made  up  of  living  cultures  has  not  proved  itself  correct. 
Carefully  killed  cultures  suffice  in  almost  all  cases.  An  example  of  the 
preparation  of  Wright's  vaccine  is  here  given. 

The  Preparation  of  a  Staphylococcus  Vaccine. 

Agar  cultures  are  grown  for  twenty-four  hours,  and  about  3  c.c.  of  sterile 
normal  saline  solution  is  added  to  each  culture.  The  growth  is  washed  off 
into  the  saline  solution  by  means  of  a  platinum  needle  or  freshly  prepared 
capillary  pipette.  The  suspension  of  bacteria  is  placed  into  a  sterile  tube, 
the  end  of  this  tube  drawn  out  in  the  blow-pipe  flame  and  sealed.  The 
drawn  out  portion  should  be  about  2  inches  in  length  and  as  strong  as 
possible.  The  emulsion  is  now  vigorously  shaken  for  fifteen  minutes.  The 
extremity  of  the  drawn-out  tube  is  then  cut  and  a  few  drops  of  the  emulsion 
expelled  into  a  clean  watch  glass,  or  a  small  part  of  the  drawn-off  end  is  cut 
off  so  that  a  portion  of  the  emulsion  is  still  contained  within  it.  The  tube  is 
resealed,  and  then  submerged  in  water  and  kept  at  60°  for  one  hour.  This 
usually  suffices  to  kill  the  bacteria. 

The  small  amount  placed  into  the  watch  glass  or  in  the  capillary  test- 
tube  serves  for  the  standardization,  which  is  carried  out  as  follows:  A 
pipette  and  rubber  bulb  as  prepared  for  the  opsonic-index  test,  is  also  used 
here.  A  volume  of  freshly  drawn  blood  of  known  corpuscular  content,  best 
taken  from  the  worker's  own  finger,  and  an  equal  unit  volume  of  bacterial 
emulsion  is  mixed  thoroughly  with  six  or  seven  volumes  of  i  1/2  per  cent,  of 
citrate  solution;  several  even  films  which  may  be  fairly  thick,  are  then 


1 88  PHAGOCYTOSIS    OPSONINS  AND    BACTERIOTROPINS. 

made  by  means  of  the  ordinary  edge  of  a  slide,  and  stained  with  carbol- 
thionin,  Leishman's  or  Jenner's  stain. 

The  entire  smear  is  divided  up  (with  a  blue  grease  pencil)  into  eight  equal 
subdivisions,  by  one  transverse  line  drawn  parallel  to  the  long  diameter  of 
the  slide  at  its  middle  and  five  vertical  lines,  one  at  each  edge  of  the  smear, 
one  in  the  center  and  one  equally  distant  between  the  edge  and  the  central 
line.  It  is  also  advantageous  to  employ  an  eye  piece,  the  field  of  which  has 
been  divided  or  made  very  much  smaller  by  the  insertion  of  a  small  paper 
screen  with  a  small  central  opening  representing  the  size  of  the  desired  field. 
Five  or  six  fields  are  then  counted  in  each  of  the  eight  subdivided  areas. 
The  number  of  red  blood  cells  seen  in  each  field  are  enumerated  in  one 
vertical  column,  the  number  of  organisms  in  the  same  field  in  another  column. 
In  this  manner  an  average  of  the  entire  slide  is  obtained. 

By  means  of  a  simple  proportional  sum,  the  number  of  bacteria  per 
cubic  centimeter  of  emulsion  is  estimated,  e.g.,  the  number  of  red  blood  cells 
counted  is  850  and  the  number  of  bacteria  1020.  The  red  blood  corpuscles 
used  in  the  standardization  are  known  to  number  5,000,000  to  a  cubic 
millimeter  or  5,000  million  to  a  cubic  centimeter;  therefore  the  number  of 
bacteria  to  a  cubic  centimeter  of  the  unknown  emulsion  is  expressed  as 
follows. 

850  :  1020  :  :  5,000,000,000  :  No.  of  bacteria  per  c.c.  of  emulsion, 
.'.6,000,000,000  =  the  number  of  bacteria  per  c.c.  of  emulsion. 

After  the  emulsion  has  been  heated  for  one  hour,  the  tube  is  unsealed  and 
a  drop  is  expressed  into  an  agar  culture  tube  which  is  incubated  for  twenty- 
four  hours  to  demonstrate  whether  the  emulsion  is  sterile  or  not.  At  the  end 
of  this  time,  if  a  growth  is  observed,  the  emulsion  must  be  heated  again  for 
one  hour  at  60°  C.  and  its  sterility  again  tested  for. 

Proper  dilution  of  the  emulsion  is  next  undertaken.  Small  bottles 
containing  25  c.c.  of  1/2  per  cent,  carbolic  acid  in  sterile  saline  are  aseptic- 
ally  closed  with  rubber  caps;  for  example,  it  is  desirable  to  make  up  these 
25  c.c.  with  staphylococcus  vaccine  so  that  each  cubic  centimeter  contains 
500  million  bacteria,  then 

(desired  amt.  to  each  c.c.) 
500,000,000X25     (No.  of  c.c.    desired) 


6,000,000,000  (dose  of  original  emulsion) 

2.08  c.c.  or  approximately  2  c.c.  of  the  original  emulsion  must  be  added  to 
the  25  c.c.  (to  be  exact  23  c.c.)  to  make  up  the  desired  dilution. 

The  rubber  cap  is  finally  coated  with  melted  paraffin  wax. 

For  stock  vaccines  it  is  best  to  make  up  the  different  vaccines  in  the 
following  concentrations : 

i.  Staphylococcus  vaccine — prepared  from  various  strains  of  staphy- 


WRIGHT'S  VACCINE  TREATMENT.  189 

lococcus,  aureus,  citreus,  and  albus,  in  three  concentrations:  1000  million, 
500  million  and  100  million,  to  the  c.c. 

2.  Streptococcus  vaccine  in  20  m.  10  m.  and  5  m.  concentrations.     Since 
the  streptococcus  grows  very  sparingly,  cultures  of  two  or  three  days  growth 
may  have  to  be  employed  for  the  preparation  of  a  vaccine,  and  even  then  it 
may  be  necessary  to  use  one  broth  culture  instead  of  sterile  salt  solution  to 
emulsify  the  agar  cultures.     On  standardizing  such  thin  vaccines  it  is  fre- 
quently necessary  to  take  one  volume  of  blood  to  two,  three,  or  even  more 
volumes  of  emulsion  and  then  calculate  accordingly. 

3.  Acne  vaccine  in  20  m.,  10  m.  and  8  m. 

4.  Mixed  acne  in  20  m.  acne  and  500  m.  staphylococcus. 

5.  Gonococcus  vaccine  in  50  m.  and  5  m.     Gonococcus  vaccines  are 
best  employed  as  autogenous  vaccines. 

6.  Typhoid  vaccine  in  1000  m.  and  2000  m.  for  prophylactic  inoculation. 

7.  Colon  vaccine  in  25  m.,  10  m.,  5  m.     Vaccines  of  coliform  organisms 
are  very  easily  emulsified;  as  a  rule  they  should  not  be  older  than  twelve 
hours  and  not  be  sterilized  for  more  than  three  quarters  of  an  hour. 

With  the  exception  of  the  staphylococcus  vaccines,  it  is  advisable  not  to 
use  stock  vaccines,  but  autogenous  vaccines,  i.e.,  vaccines  made  from  the 
specific  strain  of  bacteria  causing  the  infection  to  be  treated.  It  is  very 
important  to  isolate  the  supposed  pathogenic  organism  from  the  innocuous 
or  less  pathogenic  bacteria  contaminating  or  complicating  the  infection. 

In  tuberculosis  Wright  employs  a  dilution  of  Koch  tuberculin  (T.  R.). 
Recently  he  has  prepared  a  tubercle  bacillus  vaccine  in  the  same  way  as  the 
other  bacterial  vaccines. 

The  initial  dosage  varies  with  the  different  vaccines,  but  should  in  general 
be  about  100  to  500  million  of  staphylococci  where  one  may  go  as  high  as 
2,500  or  even  5,000  millions. 

In  colon,  streptococcus,  gonococcus  and  acne,  doses  of  i  to  3  million 
should  be  used  at  the  beginning  and  then  gradually  increased. 

In  tuberculosis  Wright  starts  with  the  T.  R.  in  dilution  equivalent  to 
about  i/iooo  mg.  of  the  dry  tuberculin  substance  and  this  is  increased  to 
about  1/600  mg. 

Wright  cites  two  general  rules  to  be  observed  in  the  therapy  of  infectious  diseases. 

1.  In  all  cases  where  the  normal  antibacterial  power  of  the  blood  has  been  lowered, 
immunization  is  indicated. 

2.  Whenever  the  blood  possesses  strongly  active  curative  powers,  an  increased  blood 
supply  to  the  infected  part  should  be  attempted  in  order  that  the  antibacterial  elements 
of  the  blood  and  leucocytes  might  display  their  effect.     In  such  cases  the  production  of 
hyperemia    is  particularly  of    help.     Similarly,   massage    and  other  such  therapeutic 
measures  can  be  useful. 

The  therapeutic  value  of  auto-inoculation  is  very  slight  and  should  not  be  encouraged, 
as  in  this  way  the  exact  dosage  cannot  be  followed  out. 

Wright  has  employed  these  vaccines  in  staphylo,  strepto,  and  gonococcus  infections, 


PHAGOCYTOSIS    OPSONINS  AND    BACTERIOTROPINS. 

as  well  as  in  coli  infections,  tuberculosis,  malta  fever  and  carcinoma  where  injections 
of  the  bacillus  neoformans  Doyen  were  given. 

From  a  critical  review  of  the  cases  published,  which  were  treated  with 
vaccines  by  Wright  and  his  fellow  workers,  one  certain  conclusion  can  be 
reached;  namely,  that  given  an  infection,  inoculations  with  small  doses  of  the 
respective  dead  or  extracted  homologous  bacteria,  will  result  in  a  therapeutic 
immunization.  Although  Koch  had  advanced  the  same  principle  for  the 
treatment  of  tuberculosis,  it  is  Wright  who.  first  recognized  the  general 
application  of  this  form  of  immunity.  Furthermore,  by  means  of  his  opsonic 
studies,  he  was  able  to  prove  that  by  the  injection  of  even  the  minutest  doses, 
for  example  1/1,000,000  c.c.  of  tuberculin,  immune  reactions  are  incited. 

In  spite  of  this  finding,  investigators  are  still  at  variance  over  the  question, 
and  two  camps  exist:  one  of  which  believes  that  the  ideal  treatment  of 
tuberculosis  consists  in  the  repetition  of  the  small  doses;  the  other,  that  the 
best  results  are  obtained  by  gradually  increasing  the  dose  of  tuberculin 
until  very  large  doses  are  administered.  Citron  has  found  the  latter  course 
more  satisfactory. 

Since,  as  is  known,  tuberculin  is  one  of  the  harmful  agents  in  tubercu- 
losis infections,  it  seems  more  advantageous  to  get  the  patient,  if  possible, 
into  such  a  condition  where  he  is  able  to  neutralize  large  doses  of  tuberculin 
rather  than  to  have  him  at  a  stage  where  even  moderate  doses  suffice  to  give 
a  reaction. 

Other  questions  of  importance  in  the  vaccine  therapy  are:  first,  whether 
any  parallelism  exists  between  the  increase  in  opsonic  index  and  improve- 
ment in  the  clinical  manifestations;  second,  whether  the  opsonic  index  must 
necessarily  be  used  as  a  guide  in  vaccine  treatment. 

As  to  the  first,  Wright  has  pointed  out  in  numerous  cases  on  record,  that 
exact  study  has  proved  that  such  parallelism  exists.  This  fact  is  probably 
correct  in  the  majority  of  instances,  but  it  cannot  be  considered  as  an  infallible 
rule,  inasmuch  as  the  formation  of  opsonins  is  only  one  of  a  great  number  of 
factors  in  the  complicated  process  of  healing,  and  consequently  one  should 
not  be  surprised  when  in  some  instances  in  spite  of  a  rising  opsonic  index, 
the  patient's  clinical  condition  becomes  worse,  and  reversely  where  improve- 
ment occurs  although  the  opsonic  index  does  not  change. 

Accordingly,  the  value  of  the  opsonic  index  during  the  course  of  treatment 
becomes  secondary  in  importance  to  the  exact  clinical  observation  of  the  case. 
Wright  and  his  school  have  shown  that  certain  bad  effects  may  follow  from 
the  injection  when  performed  during  the  negative  phase.  With  the  use 
of  small  doses  the  negative  phase  becomes  short — only  one  day  or  even  less; 
accordingly  it  is  very  probable  that  this  state  is  entirely  passed  when  an 
injection  is  repeated  on  the  fifth  to  eighth  day. 

The  tuberculin  therapy  at  the  Kraus  clinic  is  conducted  on  this  principle, 
without  estimation  of  the  opsonic  index.  And  yet,  no  harmful  effects  have 


WRIGHT'S  VACCINE  TREATMENT.  191 

ever  been  noted;  while  general  improvement,  as  increase  in  weight,  diminu- 
tion in  temperature  and  cessation  of  cough,  are  constantly  observed.  It 
would  be  illogical  to  neglect  these  clinical  data  and  give  preference  to  the 
hypothetical  action  of  opsonins  as  a  guide  in  treatment. 

It  seems  that  Wright  himself  does  not  insist  as  strongly  as  before  upon 
the  determination  of  the  opsonic  index.  One  of  his  assistants,  Matthews, 
has  recently  made  the  statement  that  in  a  great  number  of  cases  the  deter- 
mination of  the  opsonic  index  is  entirely  out  of  the  question.  If  the  choice 
between  injections  without  estimation  of  said  index  and  entire  omission  of 
inoculation  should  arise,  therapeutic  inoculation  without  the  index  is  by  all 
means  indicated.  Although  it  is  almost  a  general  tendency  at  present  to 
omit  the  opsonic  index  in  the  treatment  of  staphylococcus  infections,  this 
may  at  times  also  hold  good  in  tubercle,  gonococcus  and  streptococcus 
infections  as  well  as  in  prophylactic  typhoid  inoculations. 


CHAPTER  XVI. 

PASSIVE  IMMUNIZATION  (SERUM  THERAPY).     BACTERIOLYTIC  SERA.     SERUM 
SICKNESS.    ANAPHYLAXIS.     SPECIAL  SERUM  THERAPY. 

In  the  former  chapters  it  was  learned  that  during  active  immunization 
specific  protective  bodies  were  formed  which  circulate  in  the  blood  and  can, 
by  means  of  the  serum  be  transferred  to  another  organism.  By  animal 
experimentation  it  was  further  found  that  such  bodies  exert  this  protection 
against  fatal  intoxication  or  infection  in  various  ways;  thus,  as  antitoxins 
and  antiaggressins  they  neutralize  toxic  poisons  and  aggressins;  as  bacterio- 
lysins  they  bring  about  lysis  of  the  bacteria;  while  as  bacteriotropins  they 
prepare  the  bacteria  for  phagocytosis.  The  defending  qualities  of  such  a 
transferred  serum  is  evident  not  only  if  the  infection  is  incited  at  the  same 
time  as,  or  a  short  period  after  the  serum  is  given,  but  in  numerous  instances 
curative  effects  are  observed  if  the  serum  is  given  even  after  infection  has 
already  taken  place. 

Of  all  sera,  those  with  antitoxic  properties  have  met  the  greatest  suc- 
cess in  therapeutic  application.  They  have  already  been  referred  to  in 
their  respective  chapters. 

The  efficiency  of  the  pure  bacteriolytic  sera  on  the  other  hand  has  been  disap- 
pointing. The  reasons  given  for  this  lack  of  curative  action  is,  in  the  first 
place,  the  inability  of  bacteriolytic  serum  to  neutralize  the  endotoxins. 

Pfeiffer's  experiment  revealed  that  if  the  number  of  bacteria  exceeded  a  certain 
limit,  then  in  spite  of  bacteriolysis,  death  of  the  animal  takes  place.  This  was  explained 
by  the  existence  of  endotoxins.  By  bacteriolysis  the  endotoxins  previously  found 
within  the  bacteria  are  liberated  and  thus  get  a  chance  to  become  toxic. 

The  aim  therefore,  was  to  produce  antiendotoxic  sera.  This  was,  however,  precluded 
from  materializing  by  the  erroneous  view  of  Wolff- Eisner  who  claimed  that  it  was  impos- 
sible to  immunize  against  endotoxin. 

Numerous  methods  have  been  advocated  for  the  liberation  of  these  endotoxins: 
maceration  of  bacteria,  exposure  to  very  low  temperature,  admixture  with  chemical  sub- 
stances which  would  dissolve  the  outer  capsule,  ferment  digestion,  growth  upon  certain 
culture  media,  etc.  At  the  present  day,  there  is  absolutely  no  doubt  that  the  bacterial 
bodies  contain  poisonous  substances  against  which  it  is  difficult  and  to  a  certain  degree 
impossible  to  attain  an  immunity. 

Whether  one  should  adhere  to  the  old  idea  and  apply  to  these  the  term,  endotoxin, 
or  include  therein  the  class  of  true  toxins  with  the  only  difference  that  they  are  not  secreted 
but  contained  within  the  bacterial  body  and  therefore  more  difficult  to  isolate,  is  purely  a 
question  of  theoretical  importance. 

192 


AGGRESSIN.  193 

Another  cause  for  the  therapeutic  failure  of  bacteriolytk  serum,  is  given 
by  Bail  and  his  school,  as  the  lack  of  its  antiaggressin  action.  This  applies 
only  to  the  cases  in  which  the  bacteriolytic  serum  was  produced  by  immuniza- 
tion with  dead  bacteria. 

When  live  bacteria  are  used,  this  objection  is  not  to  be  considered,  as 
according  to  the  experiments  of  Wassermann  and  Citron,  "aggressin"  is 
nothing  more  than  the  immunizing  substance  of  the  living  bacteria.  As  for 
the  structure  of  the  antiaggressins,  the  author  was  able  to  show  that  like  the 
bacteriolysins,  they  are  amboceptors  which  bind  complement. 

Artificial  aqueous  extracts  of  living  bacteria  belonging  to  the  class  of 
half  parasites  made  according  to  the  method  of  Wassermann  and  Citron, 
contain  the  endotoxin  as  well  as  the  aggressin.  Such  artificial  aggressins, 
therefore,  represent  ideal  antigens.  The  sera  produced  by  their  injection 
contain  but  few  bacteriolytic  bodies  and  a  very  large  number  of  amboceptors, 
easily  demonstrable  by  the  Bordet-Gengou  reaction. 

Wassermann  explains  the  lack  of  therapeutic  efficiency  on  the  part  of  the 
bacteriolytic  sera,  by  the  absence  of  complement  of  the  organism,  as  well  as 
by  the  inability  of  human  complement  to  fit  all  animal  amboceptors.  As  is 
known,  amboceptors  increase  during  immunization  while  the  complement 
content  remains  the  same.  But  since  amboceptors  without  complement 
remain  inactive,  even  a  very  strong  serum  may  only  be  slightly  effective, 
depending  upon  the  amount  of  existing  complement.  If  too  many  ambo- 
ceptors are  injected,  the  serum  may  become  entirely  powerless  due  to  a 
phenomenon  similar  to  Neisser  and  Wechsberg's  complement  deviation. 
Wassermann  advises  therefore  the  addition  of  complement  to  a  serum 
before  its  injection,  in  order  to  activate  it.  This  suggestion  has  not  been 
widely  adopted  in  practice. 

It  is  for  a  similar  reason,  that  the  classical  experiment  of  bacteriolysis 
is  so  beautifully  demonstrable  in  the  guinea-pig's  peritoneal  cavity,  an 
area  relatively  poor  in  cells,  while  this  phenomenon  is  incomplete  and 
replaced  by  phagocytosis  when  occurring  in  the  blood,  inner  organs,  and  sub- 
cutaneous connective  tissue.  It  is  in  this  connection  that  Metschnikoff  and 
his  followers  see  the  main  reason  for  the  failure  of  the  therapeutic  activity 
of  bacteriolytic  sera. 

An  additional  impediment  is  offered  by  the  wide  differences  which  exist 
between  the  numerous  strains  of  the  same  bacterium.  This  may  be  so 
marked  that  an  immune  serum  produced  with  one  strain  will  enfold  no  pro- 
tection against  a  different  strain  of  the  same  bacterium.  It  is  now  overcome 
to  a  certain  extent  by  immunization  with  as  many  different  strains  of  the 
same  bacterium  as  possible  (polyvalent  sera). 

Cultures  grown  upon  artificial  media  for  a  very  long  time,  adapt  them- 
selves entirely  to  their  new  surroundings  and  frequently  lose  some  of  their 
biological  characteristics,  e.g.,  virulence.  If  the  culture  is  then  inoculated 
13 


194  PASSIVE   IMMUNIZATION. 

into  an  animal,  the  virulence  is  usually  increased  thereby  only  for  the  respec- 
tive animal  species,  but  may  at  the  same  time  be  lowered  for  man. 

Many  authors,  therefore,  employ  for  the  production  of  immune  sera  only 
virulent  strains  of  bacteria  freshly  isolated  from  man. 

In  spite  of  all  the  above  considerations,  the  fact  still  remains  that  most 
immune  sera  excepting  those  of  the  cholera,  typhoid,  and  paratyphoid 
bacteria,  show  no  bacteriolytic  tendencies  even  under  the  most  favorable 
circumstances;  but  by  means  of  their  amboceptors  they  fix  free  complement 
and  with  the  aid  of  bacteriotropins,  stimulate  phagocytosis. 

Whether  complement  fixation  is  at  all  to  be  considered  as  a  protective 
phenomenon,  cannot  with  the  presently  existing  evidence  be  definitely 
decided. 

Conditions  are  much  more  favorable  as  far  as  the  bacteriotropins  are 
concerned.  Active  phagocytosis  is  always  an  expression  of  good  resistance 
power.  It  is  not  necessary  for  the  leucocytes  to  digest  the  bacteria;  it  is 
amply  sufficient  if  a  protective  wall  of  these  cells  is  formed  (Ribbert, 
Citron,  Gruber);  moreover  they  can  neutralize  the  bacterial  poisons.  In 
this  connection  it  must  always  be  borne  in  mind  that  phagocytosis  by  no 
means  necessitates  the  death  of  bacteria. 

Granting,  however,  that  all  the  above  requirements  have  been  fulfilled 
and  a  suitable  serum  has  actually  been  produced,  will  such  a  serum  always 
be  effective,  or  are  there  any  other  causes  which  may  interfere  with  its  good 
results?  In  order  to  answer  this,  the  infectious  diseases  must  be  divided 
into  acute  and  chronic.  With  the  first  class,  success  is  quite  assured  as  long 
as  it  is  possible  to  bring  sufficient  amounts  of  the  active  serum  substances 
into  direct  contact  with  the  bacteria.  In  meningeal  infections,  intraspinal 
injections  may  have  to  be  adopted.  It  is  difficult,  however,  in  cases  of 
this  nature  to  judge  definitely  whether  the  serum  therapy  was  really  the 
effective  agent,  inasmuch  as  diseases  like  erysipelas,  meningitis,  pneu- 
monia, etc.,  are  self  limited,  lasting  for  a  period  of  time  and  then  subsiding 
of  their  own  accord. 

With  the  chronic  infections,  on  the  other  hand  (especially  tuberculosis), 
serum  therapy  has  a  new  difficulty  to  overcome.  As  a  result  of  the  long 
duration  of  the  disease,  it  is  naturally  impossible  by  means  of  a  single  injec- 
tion to  introduce  sufficient  curative  bodies,  as  can  be  accomplished  in  diph- 
theria, for  example.  It  is  necessary,  therefore,  to  repeat  the  injections  for  a 
long  period  of  time.  Under  such  conditions  the  human  organism  produces 
antibodies  against  the  foreign  proteid,  perhaps  even  against  the  curative 
substances  in  the  serum  (antiamboceptors) .  In  both  instances  the  desired 
effect  of  the  serum  is  lost. 

The  interaction  between  the  injected  serum  and  the  bodies  produced  by 
the  organism  immunizing  itself  against  it  can  manifest  itself  in  various  clin- 
ical symptoms  known  as  the  " hypersusceptibility "  reaction,  or  the  "serum 


THE    ARTHUS    PHENOMENON.  195 

sickness,"  carefully  studied  by  v.  Pirquet  and  Schick.  The  evidences  of 
serum  sickness  are  numerous.  Those  present  most  frequently  are  fever, 
skin  eruptions,  swelling  of  the  joints,  glandular  enlargement  and  edema. 

These  symptoms  may  follow  even  the  very  first  injection  of  serum.  They  develop 
as  a  rule,  after  an  incubation  period  of  eight  to  ten  days;  slight  reddening  at  the 
point  of  injection  accompanied  by  moderate  swelling  of  the  regional  lymph  glands, 
appear  as  prodromal  manifestations. 

The  general  condition  of  the  patient  is  generally  only  very  little  disturbed,  in  spite  of 
the  frequently  associated  high  fever.  Still  there  are  instances,  especially  after  the  intro- 
duction of  large  amounts  of  serum,  where  the  symptoms  continue  for  about  four  to  five 
weeks  and  then  lead  to  severe  disturbances. 

The  associated  skin  eruptions  above  mentioned,  are  usually  of  the  type  of  an  urti- 
caria; although  Hartung  describes  rashes  simulating  scarlet  and  measles. 

As  the  most  positive  symptoms  of  serum  sickness,  v.  Pirquet  and  Schick 
consider  the  following: 

1.  The  occurrence  of  the  exanthema  seven  to  fourteen  days  after  injection. 

2.  First  appearance  of  the  rash  around  the  point  of  injection. 

3.  Regional  enlargement  of  the  lymph  glands. 

4.  Complete  absence  of  any  changes  in  the  mucous  membranes. 

Measles  is  excluded  by  the  non-presence  of  Koplik  spots,  coryza,  and  conjunctivitis. 
In  scarlet  fever  the  following  symptoms  help  to  exclude  serum  sickness: 

1.  Initial  vomiting. 

2.  Occurrence  of  angina  before  or  at  the  same  time  as  the  exanthema. 

3.  High  fever. 

4.  The  simultaneous  existence  of  the  infection  among  others  in  the  hospital  or  neigh- 
borhood. 

If  the  serum  disease  does  not  arise  after  the  first,  but  after  a  later  injection,  it  is  char- 
acterized by  the  absence  of,  or  very  marked  diminution  in  the  length  of  the  period  of 
incubation,  and  in  addition  by  increased  severity  of  the  symptoms. 

Serum  sickness  belongs  to  a  group  of  conditions  designated  by 
Anaphylaxis.  the  terms  "  anaphylactic "  or  "  hypersusceptibility "  phenom- 
ena.    The  subject  of  anaphylaxis  is  one  of  present  interest 
and  its  importance  is  manifest  not  only  in  serum  sickness  and  in  the  tuber- 
culin reaction,  but  in  a  great  number  of  previously  unexplained  clinical 
occurrences.     Only  few  of  the  most  important  experimental  observations 
upon  which  this  study  is  based,  can  here  be  reviewed.     Those  of  Arthus 
and  Theobald  Smith  deserve  special  consideration. 

The  Arthus  Phenomenon. 

If  a  rabbit  is  injected  subcutaneously  with  horse's  serum  at  intervals  of  six  days,  a 
soft  infiltrate  which  remains  for  two  to  three  days  appears  at  the  site  of  injection  after  the 
fourth  inoculation,  a  harder  infiltration  which  continues  for  a  longer  period  of  time  after 
the  fifth  inoculation,  and  gangrene  after  the  sixth  or  seventh.  A  rabbit  injected  sub- 
cutaneously for  a  long  period  of  time,  on  receiving  an  intravenous  inoculation  of  horse's 
serum,  may  die  with  severe  general  symptoms  several  minutes  after  the  latter  injection. 


196  PASSIVE   IMMUNIZATION. 

The  Theobald  Smith  Phenomenon. 

Theobald  Smith  observed  that  guinea-pigs  injected  with  neutral  mixtures  of  diphtheria 
toxin  and  horse's  antitoxic  serum  would  be  killed  if  after  an  interval  of  several  weeks  they 
were  given  a  subcutaneous  injection  of  normal  horse's  serum  (several  cubic  centimeters). 

Otto  and  others  showed  that  both  of  these  phenomena,  above  described,  are  identical 
in  their  principle;  thus,  that  of  Arthus  can  be  likewise  induced  after  a  single  injection  of 
horse's  serum  if  the  first  dose  is  small,  and  if  the  interval  between  the  first  and  second 
inoculation  is  sufficiently  long  (about  three  weeks  or  more). 

Anaphylaxis  is  specific;  that  is  the  animals  made  anaphylactic  against  horse's  serum 
will  produce  a  reaction  only,  when  subsequently  injected  with  horse's  serum  and  not 
when  any  other,  like  bovine  serum  is  used.  Even  a  single  injection  of  0.001-0.004  c.c. 
of  horse's  serum  suffices  according  to  Rosenau  and  Anderson,  to  produce  anaphylaxis  in  a 
guinea-pig.  The  greater  the  amount  of  serum  given  at  the  first  inoculation,  the  longer  is 
the  period  which  must  elapse  before  the  onset  of  the  state  of  hypersusceptibility.  With 
doses  of  several  cubic  centimeters,  this  interval  is  two  to  three  months  in  duration. 

The  effect  of  the  second  injection  depends  largely  upon  the  method  of  its  administra- 
tion. Given  subcutaneously  or  intraperitoneally,  5  to  6  c.c.  are  required  to  bring  about 
acute  death  of  the  animal,  while  by  the  intravenous  or  intracerebral  route  fractions  of  a 
cubic  centimeter  usually  suffice. 

Animals  which  recover  from  their  anaphylactic  condition  after  the  second 

Antiana-       injection,  become  antianaphylactic,  i.e.,  they  do  not  react  to  further 

phylaxis.       injections  of  the  same  serum  or  proteid  solution.     Such  immunity  appears 
two  hours  after  the  recovery  from  the  anaphylactic  shock. 

In  order  to  prevent  anaphylaxis  in  animals,  Besredka  and  Steinhardt  advise  the  use 
of  a  very  small  amount  of  serum  for  the  second  injection,  followed  by  a  larger  dose  in 
twenty-four  hours;  or  an  injection  of  a  very  large  dose  during  the  period  of  incubation, 
best  on  about  the  eighth  day. 

Passive  Anaphylaxis. 

Anaphylaxis  like  immunity  can  be  transmitted  from  one  animal  to  another  by  means 
of  the  serum.  Passive  anaphylaxis  is  best  demonstrated  by  injecting  the  anaphylactic 
serum  subcutaneously,  followed  in  twenty-four  hours  by  the  inoculation  of  the  respective 
antigen. 

No  absolutely  decisive  explanation  has  as  yet  been  offered  for  the  anaphy- 
lactic status.  It  seems  certain,  however,  that  its  phenomena  are  closely 
associated  with  the  process  of  immunity. 

Since  the  term  immunization  usually  implies  a  beneficial  process,  while 
anaphylaxis  in  most  instances  represents  a  situation  of  an  injurious  nature, 
v.  Pirquet  recommended  the  term  "allergic"  to  designate  the  reactive 
changes  which  an  organism  generally  exhibits  after  infection  or  injection  of 
an  antigen.  The  " allergic  phenomena"  are  divided  into  those  associated 
with  diminished  susceptibility,  i.e.,  prophylaxis;  and  those  with  increased 
sensitiveness,  i.e.,  anaphylaxis. 

Besredka  adheres  to  the  view  that  the  anaphylactic  syndrome  especially  expresses  an 
insult  to  the  central  nervous  system.  He  was  able  to  show  that  susceptible  guinea-pigs 
when  etherized,  will  bear  the  second  inoculation  of  the  serum  perfectly  well. 

v.  Pirquet  and  Schick,  Friedberger  and  others,  consider  the  precipitin  action  as  the 


SPECIAL    SERUM   THERAPY.  197 

basis  for  the  anaphylactic  phenomena.  Other  etiological  factors,  also  come  into  con- 
sideration; such  as  the  increase  of  the  sessile  receptors  with  simultaneous  absence  or 
marked  diminution  of  free  antibodies,  and  the  absorption  of  the  complement  by  the  ambo- 
ceptors,  in  vivo. 

Attempts  have  been  made  to  employ  the  specificity  of  anaphylaxis  for 
diagnostic  purposes,  but  as  yet  the  results  obtained  do  not  justify  the  clinical 
consideration  of  the  methods. 

Special  Serum  Therapy. 

i.  Meningococcus  Serum. — Numerous  investigators  have  attempted  the 

Meningococ-   production  of  an  immune  serum  for  man,  among  these  Jochmann,  the 

cus  Immune    Berlin  Institute  for  infectious  diseases,   Ruppel,   Kraus,  Flexner  and 

Serum.        Jobling,    and  others.     The   sera   of    Jochmann   (Merck)   and  Ruppel 

(Hochst)  are  produced  by  immunization  of  horses  with  meningococci 
which  are  at  first  employed  in  dead,  and  later  in  live  form.  The  other  mentioned 
sera  are  attained  by  immunization  with  bacterial  extracts  or  bacterial  extracts  plus  full 
bacteria,  and  therefore  contain  agglutinins,  precipitins,  bacteriotropins,  amboceptors  and 
antiendotoxins.  It  is  difficult  to  test  the  efficiency  of  these  sera  in  animals,  as  the 
meningococci  vary  greatly  in  their  virulence  towards  them.  Jochmann  and  Ruppel 
assert  that  they  have  been  successful  in  growing  cultures  extremely  virulent  for  animals, 
which  they  employed  for  the  titration  of  the  therapeutic  value  of  the  serum.  In  the 
institute  for  infectious  diseases,  the  method  of  complement  fixation  is  employed  for 
the  titration  of  the  therapeutic  value  of  the  serum.  This  procedure  is  very  unreliable. 
The  protection  of  the  serum  in  mice  against  the  meningococcus  endotoxin  as  well  as 
the  demonstration  of  the  bacteriotropic  action  of  the  serum  is  far  more  significant. 

In  man,  the  immune  serum  is  injected  intraspinously,  after  a  quantity 
of  spinal  fluid  has  been  withdrawn  to  relieve  the  pressure.  In  adults  20  to  40 
c.c.  and  in  children  10  to  2oc.c.  are  daily  injected  until  either  clinical  improve- 
ment or  a  fatal  prognosis  becomes  manifest.  It  is  advisable  to  precede 
the  serum  inoculation  by  a  morphine  injection,  and  to  elevate  the  pelvis 
for  eight  to  twelve  hours  after  the  inoculation.  The  earlier  the  serum  therapy 
is  instituted,  the  more  favorable  are  its  results.  Subcutaneous  applications 
of  the  serum  or  employment  of  a  serum  older  than  three  months  is  absolutely 
of  no  use. 

Both  in  the  United  States  and  in  foreign  countries  the  value  of  the  serum  as  a  thera- 
peutic agent  seems  fairly  established.  In  Germany,  the  serum  is  obtained  gratis  at  the 
institute  for  infectious  diseases  at  Berlin.  The  serum  in  Switzerland  is  distributed  by 
the  serum  institute  of  Bern  (Kolle).  In  the  United  States,  Rockefeller's  Institute  in 
New  York  first  conducted  its  dispensation,  but  now  it  is  under  the  supervision  of  the 
New  York  Board  of  Health. 

Numerous  statistics  can  be  cited  exemplifying  the  good  results  of  the  serum.  The 
following  figures  given  by  Levy  describing  the  experiences  of  the  Essen  epidemic  are 
especially  instructive: 

From  the  first  of  January  until  the  first  of  November,  1907,  the  total  number  of  epi- 
demic meningitis  cases  which  occurred  in  Essen  were: 

55  Cases  with  29  Deaths  =52.72%  Mortality, 


198  PASSIVE    IMMUNIZATION. 

of  these,  treatment  was  given  outside  of  the  barracks  to 

15  cases  with  12  deaths  =80%  mortality, 
inside  the  barracks  were  treated 

40  cases  with  17  deaths  =42.5%  mortality, 
of  these 

14  cases  were  not  treated  with  serum  with  n  deaths  as  a  result 

=  78.6%  mortality, 
those  treated  with  serum  were 

23  cases  with  5  deaths  =  21.7%  mortality, 

of  these,  those  which  were  treated  only  incompletely  (subcutaneously)  and  with  insuffi- 
cient doses,  numbered 

6  cases  with  3  deaths  as  the  outcome  =50%  mortality, 
systematic  intraspinous  treatment  with  large  doses. 

17  cases  with  2  (i)  deaths  =  11. 8  (6.3)%  mortality. 

The  figures  in  parenthesis  represent  the  moribund  cases  coming  under  treatment  and  the 
percentage  which  would  result  if  these  were  not  included  in  the  calculation. 

The  experiences  with  the  serum  of  Flexner  and  Jobling  are  similarly  encouraging.  In 
a  report  of  400  cases  the  mortality  is  reported  as  lowered  from  80  per  cent,  to  20  per  cent. 

2.  Streptococcus  Immune  Sera. — The  rdle  of  the  streptococcus  in  some  diseases,  for 
example,  scarlet,  is  imperfectly  understood.  Moreover  it  has  only  been  indefinitely  es- 
tablished whether  there  are  various  groups  or  only  one  kind  of  streptococcus;  even  the 
significance  of  their  virulence  or  hemolysin  formation  is  not  clear.  Such  are  the  difficulties 
which  account  for  the  great  number  of  methods  advocated  for  the  production  of  an  im- 
mune streptococcus  serum.  The  oldest  serum  of  the  many,  is  that  of  Marmorek.  It  was 
produced  by  immunization  with  a  strain  made  highly  virulent  by  passage  through  animals. 
The  various  other  forms  of  the  sera  on  the  market  are: 

a.  Serum  Aronson  (Schering). — This  is  a  polyvalent  serum  produced  by  immuniza- 
tion of  horses  with  cultures  pathogenic  for  man;  some  strains  having  previously  been 
passed  through  animals,  others  not.     The  strength  of  the  serum  is  tested  in  mice  infected 
with  the  latter  strains., 

b.  Serum  Meyer-Ruppel  (Hochst  Farbwerke). — Horses  are  first  immunized  with  a 
strain  of  streptococcus  whose  virulence  has  been  raised  by  passage  through  horses  and 
mice;  each  horse  is  then  injected  with  a  different  strain  of  human  streptococcus.     When 
the  serum  of  each  animal  is  of  such  a  strength  that  doses  of  o.oi  to  0.0005  c-c-  protect  mice 
infected  .with  its  own  particular  strain,  the  sera  of  the  different  horses  are  mixed.     Thus 
a  polyvalent  serum  is  obtained. 

c.  Serum  Menzer  (Merck)  is  monovalent  and  produced  by  immunization  with  a  culture 
which  is  pathogenic  for  man  and  not  passed  through  animals. 

d.  Serum  Moser  is  polyvalent,  produced  by  injections  of  streptococci  from  scarlet 
fever.     The  sera  of  Menzer  and  Moser  are  not  tested  by  injections  of  white  mice.     The 
others  are.     One  cannot  strictly  rely  upon  this  method  of  serum  titration  for  its  employ- 
ment in  man.     The  virulence  of  streptococci  against  mice  and  human  beings  bears  no 
definite  relation.     A  serum  may  be  perfectly  efficient  in  mice  both  for  prophylactic  and 
therapeutic  purposes,  and  be  entirely  inactive  in  man;  also  vice  versa.     The  action  of  the 
serum  should  be  in  the  main  of  bacteriotropic  nature. 

Antistreptococcus  serum  has  been  tried  in  scarlet  fever,  puerperal  sepsis, 
erysipelas,  and  articular  rheumatism. 

Recently  complement  fixation  experiments  (Foix  and  Mallein,  Schleiss- 
ner)  have  shown  that  the  streptococci  of  scarlet  fever  can  be  definitely  sepa- 
rated from  the  other  varieties  of  these  bacteria. 


SPECIAL    SERUM    THERAPY.  199 

In  this  disease  favorable  results  have  been  observed  by  the  use  of 
Moser's  serum. 

Escherich  states  that  of  112  scarlet  fever  cases  injected,  those  receiving  the  serum  on 
the  first  and  second  days  of  their  illness  all  recovered  while  of  those  injected  later  on,  there 
was  a  high  percentage  of  mortality.  Other  authorities  have  seen  no  or  only  very  slight 
effect  from  the  serum  treatment. 

Two  hundred  cubic  centimeters  of  Moser's  serum  must  be  given  subcutaneously. 

The  treatment  of  puerperal  fever  has  been  favorably  influenced  by 
Aronson's  and  Meyer-Ruppel's  serum,  of  which  50  c.c.  are  injected  on 
several  successive  days. 

Menzer's  serum  is  said  to  serve  its  purpose  best  in  acute  and  chronic 
rheumatism  as  well  as  in  tuberculous  mixed  infections. 

In  erysipelas  all  the  different  well  known  sera  have  been  employed.  On  account  of 
the  very  variable  course  of  the  disease  it  is  difficult  to  judge  the  exact  value  of  the  serum 
employed.  In  fact,  thus  far  one  cannot  with  certainty  depend  upon  any  serum  treatment 
of  a  streptococcus  infection.  The  serious  nature  of  such  infections,  makes  every  possible 
therapeutic  measure  strongly  justifiable. 

3.  The   pneumococcic  sera  most   frequently  used  are  those  of  Pane, 
Pneumococ-    Romer  and  Merck.     Pane  immunizes  donkeys  with  highly  virulent  pneu- 
cus  Immune    mococci  and  uses  the  serum  for  the  treatment  of  pneumonia.     Several 
Sera.          Italian  investigators  record  favorable  results. 

Romer  prepares  a  polyvalent  serum  by  injecting  horses  with  different  strains  of 
pneumococcus  obtained  directly  from  man;  the  strength  of  the  serum  is  tested  in  mice. 
The  serum  is  mainly  employed  both  for  the  protection  and  cure  of  ulcus  cornea  serpens. 

The  result  according  to  Romer  depends  upon  the  very  variable  virulence  of  the  pneu- 
mococci.  The  severity  of  the  infection  in  man  is  said  to  run  parallel  with  the  virulence  in 
mice.  Romer,  therefore,  ascertains  in  every  case  of  ulcus  serpens  whether  his  serum  has 
any  protective  bodies  for  that  particular  strain  of  pneumococcus,  and  tests  the  virulence 
of  the  same. 

The  serum  can  be  injected  intravenously  and  subcutaneously,  and  in  pneumococcus 
meningitis,  intradurally.  It  is  manufactured  by  the  Hochst  Farbwerke,  in  vials  of  10 
and  20  c.c. 

A  similar  serum  is  manufactured  by  Merck.  It  is  obtained  from  horses  and  stand- 
ardized at  the  Institute  for  experimental  therapy  in  Frankfort  o/M.  so  that  o.oi  c.c.  injected 
subcutaneously  protects  a  mouse  inoculated  intraperitoneally  twenty-four  hours  later  with 
10  to  100  times  the  lethal  dose  of  a  living  pneumococcus  culture.  This  is  known  as  a 
normal  serum  and  one  cubic  centimeter  contains  one  immunity  unit  (I.  E.).  The  serum 
on  the  market  contains  20  to  40  units  per  c.c. 

In  pneumonia  200  to  400  units  are  given  subcutaneously  and  repeated  in  three  to 
four  days,  if  the  fever  does  not  subside.  As  a  prophylactic  inoculation  200  to  400 
units  are  given  to  old  people  where  a  "hypostatic"  pneumonia  is  feared.  In  ulcus 
serpens  of  the  cornea  200  to  400  units  are  employed  and  if  no  improvement  sets  in,  the 
dose  is  repeated  upon  the  third  day.  In  addition,  several  drops  of  the  serum  are 
instilled  into  the  conjunctival  sac  every  two  hours.  As  a  prophylactic  dose  in  this 
disease  100  units  suffice. 

Merck  also  prepares  a  vaccine  of  dead  pneumococci  in  doses  of  i  c.c.  which  further 
aid  in  the  treatment  of  pneumococcic  infections,  i  c.c.  of  such  dead  pneumococci  can 
be  administered  for  the  prophylaxis  of  ulcus  serpens. 

4.  Pest  Sera. — A  large  number  of  pest  sera  are  in  use. 


200  PASSIVE    IMMUNIZATION. 

a.  The  Paris  serum  (Yersin)  produced  at  Pasteur  Institute  by  immunization  of  horses 
with  dead  and  later  on  living  bacilli. 

b.  The  Bern  serum  of  Tavel  employs  the  same  principles. 

c.  Lustig's  Serum. — For  this  serum,  horses  are  immunized  with  the  pest-nucleopro- 
teids.     Pest  cultures  are  broken  up  by  i  per  cent,  of  potassium  hydroxide  and  from  this, 
by  the  addition  of  acetic  acid,  the  nucleoproteid  is  precipitated  and  then  suspended  in  salt 
solution  to  serve  as  antigen. 

d.  Serum  of  Terni-Bandi  is  prepared  by  the  immunization  of  donkeys  and  sheep  with 
natural  pest  aggressins. 

e.  Serum  of  Markl  is  supposedly  an  antitoxic  serum  prepared  by  immunization  with 
nitrates  of  old  pest  bouillon  cultures. 

All  the  above  sera  contain  agglutinins,  precipitins,  bacteriotropins  and  amboceptors; 
the  serum  of  Terni-Bandi  contains  aggressin  amboceptors,  that  of  Markl,  antiendotoxins. 

The  sera  are  tested  for  their  anti-infectious  properties  in  animals  such  as  guinea-pigs, 
rats,  mice.  Markl  also  estimates  the  toxin  neutralization  power  of  his  serum. 

The  Paris  serum  comes  either  in  dry  form  or  in  bottles  containing  20  c.c.  without  any 
preservatives.  Ten  to  20  c.c.  should  suffice  as  a  prophylactic  injection,  although  Martini 
advises  100  c.c.  at  least.  The  period  of  protection  is  short,  averaging  about  fourteen  days. 

Prophylactic  injection  is  advisable  in  those  instances  where  an  immediate  protection 
is  necessary,  like  the  inoculation  of  physicians  and  nurses  attending  pest  patients.  Under 
all  other  circumstances  either  active  immunization  or  the  simultaneous  method  of  Shiga 
should  receive  the  preference. 

For  the  treatment  of  pest  infections,  Calmette  and  Salimbeni  advise  intravenous  ad- 
ministration of  20  c.c.  and  two  subcutaneous  injections  of  40  c.c.  each — all  to  be  given  on 
the  first  day;  on  the  second  day  two  similar  subcutaneous  injections;  and  if  the  case  is  of  a 
severe  nature,  the  dose  may  be  doubled.  The  results  are  variable. 

From  comparative  studies,  it  seems  that  Lustig's  serum  is  somewhat  weaker  than  the 
Paris  serum.  The  sera  of  Terni-Bandi  and  Markl  have  not  been  sufficiently  employed, 
so  that  opinion  is  reserved. 

5.  Tuberculosis  Sera. — The  best  known  and  most  studied  are  those  of  Maragliano  and 
Marmorek. 

a.  Serum  Maragliano  is  prepared  by  Maragliano's  institute  in  Genoa  from  horses 
which  are  immunized  for  about  six  months  with  the  soluble  substances  of  tubercle  bacilli. 
The  favorable  action  of  the  serum  is  reported  on,  especially  by  Italian  authorities. 

b.  Serum  Marmorek  is  prepared  in  the  laboratory  of  Marmorek,  at  Paris-Neuilly,  by 
the  immunization  of  horses  with  the  so-called  "primitive"  tubercle  bacilli,  i.e.,  young 
tubercle  bacilli  whose  acid  fast  character  is  still  very  slight  or  entirely  absent.     When  the 
horses  have  attained  a  high  grade  of  immunity,  they  receive  injections  of  various  strains 
of  pure  cultures  of  streptococci  obtained  from  the  sputum  of  tuberculous  patients.     The 
serum  of  these  animals  is,  therefore,  antituberculous  and  at  the  same  time  polyvalent 
antistreptococcic  (a  double  serum),  serving  against  the  mixed  infections. 

This  serum  is  administered  daily,  either  subcutaneously  5  to  10  c.c.  or  per  rectum 
20  c.c.  The  latter  form  is  more  advisable  for  the  sake  of  preventing  anaphylaxis.  Citron 
has  found  the  serum  entirely  harmless,  the  bad  effects  described  by  some  being  probably 
due  to  the  idiosyncrasy  of  patients  against  foreign  sera.  The  most  favorable  results  have 
been  claimed  as  found  in  localized  bone  and  joint  tuberculosis  and  in  the  incipient  stages 
of  pulmonary  tuberculosis.  Especial  consideration  of  the  serum  should  be  given  in 
those  patients  who  evince  persistent  temperature  or  the  very  severe  but  not  hopeless  cases, 
where  the  tuberculin  therapy  cannot  be  undertaken.  In  some  of  these  instances  very 
encouraging  results  have  been  noted. 


SPECIAL    SERUM    THERAPY.  2OI 

Occasionally  the  author  started  with  the  serum  treatment,  and  then  combined  with  it 
the  tuberculin  administration  and  finally  left  the  serum  away  entirely. 

6.  Anthrax  Sera. — Sclavo,  Deutsh,  Sobernheim   and  others  have  produced  immune 
sera  by  the  immunization  of  donkeys,  sheep  and  horses.     These  have  been  mainly  em- 
ployed in  veterinary  practice. 

In  man  the  serum  has  been  tried  only  by  Sclavo.  He  injects  30  to  40  c.c.  subcutan- 
eously  for  several  successive  days;  in  severe  infections  10  c.c.  are  administered  intra- 
venously. Two  cases  described  by  Bandi  received  150  c.c.  intravenously. 

7.  Typhoid  Immune  Sera. — The  ordinary  bacteriolytic  sera  (Tavel)  have  not  met  with 
the  desired  success  in  the  therapy  of  typhoid  fever.     Attempts  have,  therefore,  been  made 
to  produce  antiendotoxic  sera.  Chantemesse  treats  horses  for  several  years  with  bouillon 
filtrates;  Besredka  injects  first  dead  and  then  living  typhoid  bacteria  from  agar  cultures, 
Mac  Fadyen  breaks  up  the  bacteria  at  very  low  temperatures  and  thus  liberates  the  endo- 
toxin  for  purposes  of  immunization.     Kraus  and  von  Stenitzer  use  bouillon  filtrates  and 
aqueous  bacterial  extracts  as  is  likewise  done  by  Meyer-Bergell  and  Aronson.     Garbat 
and  Meyer  employ  sensitized  typhoid  bacilli,  i.e.,  bacteria  united  with  their  bacteriolytic 
amboceptors. 

Chantemesse  injects  several  drops  of  his  serum  subcutaneously.  The  action  lasts  ten 
days.  Only  occasionally  is  a  second  inoculation  necessary;  if  so,  it  must  be  much  smaller. 
His  results  have  been  good  and  have  mainly  depended  upon  an  increase  in  the  opsonic 
index. 

Meyer  and  Bergell  as  well  as  Kraus  give  20  to  50  c.c.  subcutaneously. 

8.  Cholera  Serum. — Similar  attempts  for  the  production  of  a  cholera  antiendotoxic 
serum  have  been  made.     Kraus  has  succeeded  in  obtaining  an  antitoxin  against  some 
El-Tor  vibrios  which  have  all  the  characteristics  of  true  cholera  vibrios. 

The  experiments  with  Kraus'  serum,  and  Kolle's  serum  (Bern  Institute),  at  present 
being  conducted  in  Russia,  seem  to  be  favorable. 

The  serum  therapy  of  infectious  diseases  is  still  in  its  primitive  stages. 
The  contradictory  results  of  many  authors  are  to  be  associated  not  only 
with  the  variable  efficiency  of  the  sera,  but  also  with  the  method,  the  time, 
and  the  dose  chosen  for  administration. 

The  same  serum  in  the  hands  of  different  physicians  may  yield  opposite 
results.  These  subjective  sources  of  error  must  be  overcome,  or  minimized 
by  making  a  complete  and  thorough  study  of  the  effects  which  a  certain 
serum  may  have  and  actually  does  have;  here  all  the  clinical  and  laboratory 
guides  must  be  made  use  of.  Employed  in  this  manner,  serum  therapy 
will  even  at  the  present  stage  lead  to  beneficial  results. 

Wright's  motto  at  the  beginning  of  his  book  on  vaccines  "The  physician 
of  the  future  will  be  an  immunisator,"  can  justly  be  reversed  to  read,  "the 
immunisator  of  the  future  will  be  a  physician." 


Plate  1, 


Fig.  1.    Positive  v.  Pirquet  Reaction 

(Original  drawing) 


1 


Fig.  2.    Ophthalmo-reaction 

(Original  drawing) 


a)  Control  eye 


b)  Reaction  of  l°-2°  grade 


Fig.  1.    Very  strongly  positive  Wassermann  Reaction  (-j — | — | — [-) 


Fig.  2.    Strongly  positive  Wassermann  Reaction  H — \ — (-) 


Fig.  3.    Positive  Wassermann  Reaction  (++) 


Fig.  7. 
a)  after  24  hrs  b)  t 


Plate  2. 


Fig.  4.    Weakly  positive  Wassermann  Reaction  (+) 

I 


Fig.  5.    Doubtful  Wassermann  Reaction  (+) 


haemolysis  Fig.  6.    Negative  Wassermann  Reaction  (— ) 

same  speciman  shaken  up. 


SUBJECTS,  INDEX. 


(Numbers  refer  to  pages.) 


Abrin,  87,  88 

Actinomyces,  174 

Agglutination,  30,  97 

Agglutinins,  32,  33,  97-107,  197,  200 

Agglutinogen,  104 

Agglutinoids,  104 

Agglutinophore  group,  104,  147 

Aggressins,  34-42,  193 

—  artificial,  30,  36 

-  natural,  30,  34,  35,  36,  193 

Albumin  differentiation,  112-117,  142,  171-173 

Albumins,  112-117,  142 

Alexin,  120 

Amboceptors,  120,  137,  146,  147,  194,  197,  200 

Anaemia  perniciosa,  93 

Anaphylaxis,  passive,  196,  197 

Animal  bacteria,  104 

Animal  sepsis,  varieties,  22 

Ankylostoma,  153 

Anthrax,  26,  68,  156,  201 

Antiaggressin,  41,  192,  193,  200 

Antiamboceptors,  194 

Antianaphylaxis,  196 

Antibodies,  3,  4,  5,  30,  145,  147,  152,  155 

Antibody  production,  local,  59,  87,  147 

An tiendo toxin,  192,  197,  200,  201 

Antiferments,  94-96 

Antigen,  21,  22,  102,  141,  142,  149,  155,  157 

Antigenophile  group,  141 

An  tihemo  toxin,  85,  87,  93 

Antileucocyte  ferment,  95 

Antilysin,  85-87 

Antiserum,  157 

Antistaphylolysin,  85-87 

Antitoxin,  71-94,  147,  192,  196 

Antitrypsin,  94-96 

Antituberculin,  46,  142,  143,  145-148 

Aortitis,  151 

Arachnolysin,  59 

Arthus  phenomenon,  195 

Ascarides,  154 

Autoinoculation,  180,  181,  189 

—  in  tuberculosis,  179,  180 

Autumn  catarrh,  89 

Bacilli  emulsion  (Koch),  58 
Bacillus  neoformans,  Doyen,  190 
Bacterial  emulsion,  182-184 
Bacterial  extract,  34,  36-42,  157,  197 
Bacterial  filter,  16,  18 
Bacterial  precipitin,  109,  112 
Bactericidal  plate  method,  128-130 
Bacteriolysin,  31,  33,  119,  120 
Bacteriolysis,  30,  118-128,  192,  193 
Bacteriotropin,  31,  175,  194,  197,  200 
Basedow's  disease,  95 
Bee  poison,  89,  100 
Beraneck's  tuberculin,  58 


Biological  mercurial  therapy,  152-154 
Blood  cells,  washing  of,  132,  155,  158,  165 
Blood  pressure,  fall  in  diphtheria.  75 
Blood  relationship,  113 
Blood  removal,  13 

—  by  wet  cups,  13 

—  from  vein,  13 
Bothriocephalus  latus,  93 
Botulism  toxin,  79,  81,  82 
Bovovaccine,  29,  66 

Brieger's  cachexia  (carcinoma)  reaction,  94-96 

Cachexia,  94 

Capillary  pipettes,  123 

Carcinoma,  94,  190 

Casein,  96 

Castellani's  test,  103,  104 

Centrifuge  rules,  17 

Chamber  land  filter,  17 

Chicken  cholera  26,  34,  39 

Cholera  extract,  42 

Cholera  serum,  97,  98-100,  118,  119,  130,  201 

Cholera  vibrios,  30,  86,  97,  98-105,  118,  119, 

125,  127,  130 
Cholestrin,  82,  92 
Cobra  poison,  90,  91,  92 
Coli  bacilli,  30,  99,  156,  183,  185,  189 
Colubrides  poison,  89 
Complement,  119,  133,  135,  137,  138,  155,  157, 

178,  193,  194 
Complement   deviation    (Neisser-Wechsberg), 

136,  iQ3 
Complement  fixation,  30,  134,  i39,-i54 

—  technique,  155-173 
Complementoids,  134 
Complementophile  group,  120,  147 
Control  tests,  value,  5,  6,  158 
Cow-pox,  25 

Conjunctiva  reaction,  48-51,  53,  54 

Crotin,  88 

Cutaneous  reaction,  47,  48,  52,  54,  147 

Cytase,  139,  176 

Cytolysin,  138 

Cytophile  group,  121 

Cytotoxin,  138 

Dilutions,  18 

—  preparation  of,  18,  19,  20 
Diphtheria,  68 
Diphtheria  serum,  68-79,  196 

—  standardization,  73-76 

—  therapeutic  application,  75,  77,  192 

—  prophylactic  application,  77 
Donath  Landsteiner's  test,  93,  94 
Dysentery  antitoxin,  83-85 
Dysentery  bacilli,  30,  98,  105,  130 
Dysentery  serum,  83,  85,  105,  130 
Dysentery  toxin.  79,  83-85 


203 


204 


SUBJECTS,    INDEX. 


Echinococcus,  154,  171 
Ehrlich's  experiment,  93 
Ehrlich's  side  chain  theory,  104,  146-165 
Endocarditis  maligna,  105 
Endotoxin,  78,  125,  192,  197 
Ergophore  group,  104,  134 
Erysipelas,  194,  198 
Erythrocytes,  see  blood  cells 
Exudate,  34,  35,  123,  157 

—  removal  of,  1 23 

Fatty  acids,  93 

Febris  recurrens,  151 

Ferments,  94-96,  147 

Fever,  147 

Ficker's  diagnosticum,  99 

Filtration,  16 

Focal  reaction,  60 

Food-stuff  substitution,  113,  115 

Forensic  serum  differentiation,  113-115 

Fornet's  ring  test,  no,  in 

Fowl  plague,  34 

Frambesia,  151 

Friedberger's  position,  n,  123 

Functionating  radicle  of  cell  (biological),  146 

Glanders,  107 
Glycogen,  142 
Gonococcus  vaccine,  189 
Group  agglutination,  101-104 
.  Group  reactions,  no 
Guinea-pig  sepsis,  21 

Haptine,  147 

Haptophore  group,  104,  134,  147 
Hay  fever,  88,  89 
Helminthiasis,  154 
Hemagglutinin,  107 
Hemoglobinuria,  93 
Hemolysin,  98,  131^138,  155,  157,  198 
Hemorrhagin,  90 
Hemotoxin,  78,  79,  89-94 
Hog  cholera,  105 
Hypersusceptibility,  see 
—  anaphylaxis 

Immune  bodies,  120 
Immune  hemolysin,  131-138 
Immunity,  3 

—  absolute  and  relative,  4 

—  active,  21 

—  antiaggressin,  38 

—  antitoxic,  3,  92 

—  attained,  4,  146 
chicken  cholera,  39 

—  conception  of,  3 
diphtheria,  71-77 

—  against  hog  cholera,  4,  105 

—  in  lues,  152,  153 

—  against  snake  poison,  92 
—  swine  pest,  39,  40,  41 

—  bactericidal,  3,  38 

—  cellular,  3 

—  continued,  4 

—  genera],  4 

—  "histogene,"  3 

—  local,  4,  58,  88,  147 

—  natural,  4,  146 

—  partial,  58,  103,  104 

—  passive,  212-224 


—  "tissue,"  3 

—  transitory,  4 

Immunization,  active  principle  of,  21,  71 
—  technique,  22,  71 

—  with  aggressins,  38-42 

—  with  dead  virus,  30-33 
with  erythrocytes,  132 

with  living  virus,  22-30 

—  with  toxins,  71-77 
Inactivation,  119,  120,  122,  157 
Incubation  period,  27,  69,  79,  195,  196 
Injection,  technique,  9-12,  35 

—  intracardial,  10,  77 

—  intracerebral,  80,  195,  196 

—  intralumbar,  194,  197 

—  intramuscular,  77 

—  intraneural,  81 

—  intraperitoneal,  n,  77,  194,  196 

—  intravenous,  9,  10,  200,  201 

—  rectal,  200 

—  subcutaneous,  12,  77,  196,  200,  201 

—  subdural,  81 
Isoprecipitins,  113 

Jennerian  immunization,  25 
Jequirity  seed,  87 

Kidney  tuberculosis,  62 
Killing  of  bacteria,  30 
Klausner's  reaction,  112 
Kolles'  flasks,  36 

Laboratory  equipment,  7-9 

Law  of  multiple  proportions,  78,  146,  149 

Lecithin,  82,  89,  90,  91,  92 

Lecithin  hemo toxins,  89,  etc. 

Leprosy,  151,  174 

Leucoantifermantin,  95 

Leucocidin,  138 

Leucocytes,  4,  34,  175,  etc.,  193 

—  obtention  of,  181,  182 
Lilliputian  filter,  17 
Limes  death,  74,  75 
Limes  zero,  74,  75 

Lipoids,  see  lecithin,  100,  and  cholestrin. 
Local  formation  of  antibodies,  58,  88,  147 
LoefHer's  serum  plates,  95 
Loop  standard,  8,  19 
Lues,  see  syphilis 

—  asymptomatica,  153 
Lupus,  62 

Lyssa,  26-29,  55 

Macrocytase,  139,  176 

Macrophage,  174,  175 

Malaria,  105,  151,  152 

Mallein,  54 

Malta  fever,  106,  190 

Measles,  112,  166,  195 

Meningitis,  105,  158-161,  194,  197,  198 

—  pneumococcus,  199 
Meningococci,  30,  86,  157,  194 
Meningococcic  serum,  105,  158-161,  197,  198 
Mercurial  therapy  (biological),  151-154 
Metschnikoff's  experiment,  125 
Microcytase,  139,  176 

Mouse-typhoid  bacilli,  105 
Multipartial  sera,  103,  193 
Mushroom  poison,  89 


SUBJECTS,    INDEX. 


205 


Nastin,  66,  67 
Negative  phase,  72,  178 
Nephrotoxin,  138 
Neurotoxin,  79,  80,  90,  92,  138 
Neutral  red,  176 
New  tuberculin,  58,  etc.,  63-66 
Non-binding  doses,  143-145 
Normal  bacteriolysins,  125 
Normal  curative  serum,  73 
Normal  hemolysin,  125 
Normal  loop,  10,  19,  20 
Normal  toxins,  73 

Ointment  reaction,  Moro,  48 
Oleic  acid,  91-93 
Ophthalmo  reaction,  48-51,  147 
Opsonic  index,  177-187 
Opsonins,  176-192 
Opsonizer,  183 
Original  tuberculin,  old,  56 
Ozena  bacilli,  no 

Paralysis,  progressive,  149,  150,  152 
Parasites,  23,  34,  193 
-  half,  23,  34,  193 

—  total,  23,  34 

Paratyphoid  bacilli,  30,  101-105,  127 

Paroxysmal  hemoglobinuria,  93 

Partial  agglutinins,  102,  103 

Partial  aggressins,  57 

Partial  immunization,  58,  102,  103 

Pathogenicity,  23 

Pernicious  anemia,  93 

Pest,  106,  156,  199,  200 

Pest  sera,  101,  199,  200 

Pfeiffer's  phenomenon,  118,  121-127 

Phagocytosis,  175,  etc.,  193 

—  during  artificial  immunity,  175-189 

—  during  natural  immunity,  4 
Phagolysis,  175 
Phrynolysin,  89 
Phytotoxin,  87,  88,  89 
Pirquet's  reaction,  47-48,  52 
Pneumococci,  30,  199 
Pneumococcic  sera,  199 
Pneumonia,  95,  194,  199 
Pneumonia  bacilli,  no 
Pollantin,  88 

Pollen  poison,  87,  88,  89 

Polyvalent  sera,  103,  193 

Porges  reaction,  in 

Positive  phase,  178 

Precipitation,  108-117 

Precipitinogen,  108,  no,  etc. 

Precipitinoids,  109 

Precipitinophore  group,  147 

Precipitins,  108-117,  147 

Prognostic  employment  of  autoinoculation,  181 

—  of  the  lues  reaction,  151 
Prophylactic  inoculations  in 

—  cholera,  42 

—  lyssa,  26,  27 

—  small-pox,  26 

—  swine  erysipelas,  30 

—  typhoid,  31,  32,  42 
Prophylaxis  in  diphtheria,  77 

—  in  dysentery,  84,  85 

—  against  hay  fever,  89 

—  in  pest,  200 

—  in  tetanus,  81 


—  in  ulcus  corneae,  200 

Proteid  differentiation,  112-117,  I7I~I73 
Proteids,  112-117,  J42 
Pseudoagglutination,  100 
Psychoreaction,  92 
Puerperal  sepsis,  198 
Pukal  filter,  17 

Rabbit  sepsis,  21 

Rat  trypanosomiasis,  22 

"Reagine,"  151,  etc. 

Receptors,  120, 122,  134,  138,  146-149,  193, 194 

Reichel  filter,  17 

Rheumatic  fever,  198 

Rhinoscleroma  bacilli,  no 

Ricin,  87 

Ring  test,  no 

Saprophytes,  23,  28 
Scarlet,  in,  151,  157,  166,  195 
Scorpion  poison,  89-92 
Seiden  pepton,  142 
Sensitized  bacteria,  29,  65,  66 
Sepsis,  105,  198 
Serum,  color,  15 

—  obtaining  of,  12,  13,  73 

—  preservation,  15,  16,  73 

Serum  diagnosis,  see  under  individual  infec- 
tious diseases 

—  in  meningitis,  158-161 

—  in  syphilis,  in,  112,  148,  154,  162-170 
Serum  sickness,  77,  195-197 

Serum  therapy,  79,  192-201 

—  in  anthrax,  201 

—  in  cholera,  201 

—  in  diphtheria,  76,  77 

—  in  erysipelas,  198 

—  in  hay  fever,  88,  89 

—  in  meningitis,  197,  198 

—  in  pest,  199,  200 

—  in  pneumonia,  199 

—  in  rheumatism,  198 

—  in  scarlet  fever,  199 

—  in  sepsis,  198 

—  in  streptococcic  infections,  198,  199 

—  in  tetanus,  81 

—  in  tuberculosis,  200,  201 

—  in  typhoid  fever,  201 

—  in  ulcus  serpens,  199 
Side  chain  theory,  146,  147 
"Simultaneous  method,"  30,  71,  209 
Small- pox,  25 

Smith's  phenomenon,  196 
Snake  poison,  89-92 

—  serum,  92 

Specificity,  5,  97,  102,  112,  116,  121 

Spermotoxin,  138 

Spider  poison,  89 

Spinal  fluid,  158-161 

Spreader,  185 

Staphylococci,  30,  177,  178,  182,  187,  188 

—  vaccine,  187 
Staphylohemo toxin,  85,  86 
Staphylolysin,  79,  85-88 
Strauss  canula,  13 

Street  virus,  26 
Streptococci,  30,  157,  184 

—  sera,  198,  199,  200 

—  vaccine,  189,  200 


206 


SUBJECTS,    INDEX. 


Substance  sensibilisatrice,  120,  see  amboceptor, 

bacteriolysin 

Summation  of  antigen,  143,  144,  158 
Swine  erysipelas,  30 
Swine  sepsis,  34,  39,  40,  41 
Syphilis,  in,  112,  148-154,  162-170 

—  active,  151,  152 

—  antigen,  149,  162,  163,  167 
System  control,  158 

Tabes  dorsalis,  149,  150,  152 
Tauruman,  29,  66 
Tetanolysin,  78,  80,  81 
Tetanospasmin,  79,  80,  146 
Tetanus,  79,  80 

—  cerebral,  80 

—  sine  tetano,  80 
Tetanus  antitoxin,  81 
Tetanus  toxin,  79,  80 

Thermolabile  substances  of  the  serum,  15,  16, 

1 20 

Thermoresistant  substances,  15,  101,  120,  177 
Thyroid,  95 

Titration  of  luetic  sera,  163-167 
Toad  poison,  89 
Toxins,  68-96,  134,  147 

—  action,  69 

—  definition  of,  18 

—  obtaining  of,  68-69 

—  titration  of,  70 
Toxoids,  75,  134 
Toxolipoids,  93 
Toxon,  74 

Toxophore  group,  134 
Transudate,  158 
Trichophytin,  54 
Trypanosomiasis,  151,  152 
Trypsin,  94-96 

Tubercle  bacilli,  55,  56,  57,  157,  174,  183 
Tuberculin,  43-67,  142-148,  179,  180,  195 

—  action,  58-59 

—  Beraneck's  tuberculin,  58 

—  bovine,  66 

—  diagnosis,  45~54 


—  new  tuberculin,  58,  63-65,  189,  190 

—  obtaining  of,  43 

—  old  tuberculin,  45-57,  59>  60,  63 

—  original  old  tuberculin,  57 

—  reaction,  45-54,  195 

—  theory,  142-148 

—  therapy,  55-67,  107,  142-143,  189-191,  201 

—  vacuum  tuberculin,  57 
-  watery,  57 

Tuberculosis,  92,  105,  106,  107,  194 

—  treatment  in  successive  steps,  61 

—  vaccination  in,  29,  189-191 
Tuberculosis  sera,  92,  101,  106,  107,  200,  201 
Typhoid,  128,  166,  201 

Typhoid  bacilli,  30,  86,  98-105,  in,  118,  119, 

J57.  l8S 

—  vaccine,  189 
Typhoid  extract,  42 

Typhoid  protective  inoculation,  31,  32 
Typhoid  serum,  98,  101,  118,  119,  201 

Ulcus  corneae,  200 
Urticaria,  195 

Vaccine,  after  Pasteur,  25-28,  39 

—  after  Wright,  178-181,  187-191 
Vacuum  desiccator,  15,  16 
Vacuum  tuberculin,  57 
Venopuncture,  12,  13 

Viper's  poison,  90 
Virulence,  23,  122,  193,  194,  198 
Virus  fixe,  26,  27 
Vital  staining,  176 

Wall  of  leucocytes,  194 
Wassermann's  reaction,  see  syphilis 
Watery  tuberculin,  57 
Weigert's  law,  146 
Wet  cupping  for  obtaining  blood,  13 
Wet  nurse,  examination  for  syphilis,  153 
Whooping-cough  bacilli,  157 
Widal's  test,  98,  99 

Zootoxins,  87,  89-91 


INDEX  OF  AUTHORS. 


Anderson,  80,  195 
Arloing,  106 
Arndt,  86 
Aronson,  198,  199 
Arrhenius,  79 
Arthus,  195 
Audeoud, 
Aufrecht 


"I4 
,63 


Bail,  23,  30,  33,  36,  38,  39,  40,  42,  126,  193 

Bamberg,  96 

Bandelier,  45,  63,  64 

Bandi,  200,  201 

Bassenge,  42 

Bauer,  92,  168,  169,  170 

v.  Behring,  29,  66,  71,  73,  81,  146 

Beraneck,  58 

Bergell,  201 

Berger,  48 

Berghaus,  76 

v.  Bergmann,  94 

Bertrand,  92 

Besredka,  195,  197,  201 

Bier,  14 

Blaschko,  150 

Boas,  152 

Bockenheimer,  81 

Boer,  73 

Bordet,  3,  79,  119,  120,  130,  131,  139,  141,  142, 

155,  161 
Borelli,  150 

Brieger,  42,  57,  94,  126,  168 
Bruck,  59,  85,  86,  142,  148,  149,  150,  161,  171 
Buchner,  120 

Calmette,  43,  49,  81,  92,  93,  200 

Castellani,  103,  105 

Chamberland,  30 

Chantemesse,  201 

Christian,  147 

Citron,  36,  49,  54,  57.  I4i,  142,  150,  IS1.  IO1 

Coca,  91 

Cohen,  157 

Conradi,  83 

Cornet,  101 

Courmont,  106 

Czaplewski,  8 

Denys,  57,  176 
Deutsch,  201 
Deycke,  66 
Doenitz,  77,  79,  80 
Doerr,  83,  84 
Doganoff,  48 
Donath,  93,  94 
Dopter,  84 


Douglas,  176 
Dunbar,  88 
v.  Dungern,  91,  131 
Durham,  97 

Ehrlich,  2,  71,  73,  78,  80,  93,  104,  120,  131,  134 

139,  145,  146,  147,  176 
v.  Eisler,  no 
Eppenstein,  54 
van  Ermenghem,  81 
Escherich,  199 

Ferran,  28 

Ficker,  99 

Fleischmann,  150 

Fleming,  170 

Flexner,  83,  90,  106,  195 

Foix,  157,  198 

Fornet,  no,  in 

Forssmann,  83 

Fournier,  153 

Frankel,  C.,  71 

Franz,  52 

Freemann,  179 

Friedberger,  n,  16,  123,  147,  195 

Friedmann,  29 

Fuld,  96 

Garbat,  161,  168,  169,  201 
Gengou,  3,  130,  139,  141,  142,  155 
Ghedini,  154,  171 
Goetsch,  60 
Grosz,  96 
Gruber,  97,  194 

Hecht,  170 
Helmann,  54 
Hendersen-Smith,  77 
Hirschfeld,  161 
Hogyes,  28 
Hoke,  42 
Holzmann,  92 
Huppe,  42 

Issaeff,  123 

Jaffe,  129,  130 
Jenner,  3,  25 
Jobling,  195 
Jochmann,  94,  95,  195 

Kelning,  54 
Kempner,  82 
Kikuchi,  42 
Kitasato,  71,  80 
Kitashima,  71 
Klausner,  112 


207 


208 


INDEX    OF   AUTHORS. 


Kleine,  107 

Koch,  R.,  29,  43,  45,  46,  57,  59,  66,  106 

Kolle,  29,  43,  45,  57,  59,  66,  106,  197 

Koplik,  195 

Korte,  128,  130 

Kossel,  76 

Kramer,  63 

Kraus,  F.,  149 

Kraus,  R.,  75,  78,  83,  84,  108,  161,  195,  201 

Kreibich,  112 

Kruse,  83,  106 

Kyes,  90 

Landsteiner,  93,  94,  131,  167,  169 

Laubry,  154 

Leclef,  176 

Ledermann,  150 

Leishman,  176,  188 

Lenhartz,  62 

Lesourd,  141 

Leuchs,  157,  161 

Levaditi,  150,  175 

Levy,  195 

Liefmann,  142 

Lignieres,  48 

Loewenstein,  46 

Lorenz,  30 

Lustig,  200 

Mac  Fadyen,  201 

Madsen,  72,  73,  77,  79,  82 

Mallein,  157,  198 

Maragliano,  57,  200 

Marcus,  94,  95 

Marie,  150 

Markl,  200 

Marmorek,  200 

Martin,  71 

Matthews,  191 

Mayer,  42 

Meier,  G.,  no,  in,  167 

Menzer,  198 

Merck,  99,  198,  199 

Metallnikoff,  67 

Metschnikoff,  2,  125,  126,  138,  139,  174,  176 

Meyer,  80 

Meyer,  F.,  65,  77,  198,  201 

Meyer,  K.,  94,  96 

Michaelis,  G.,  85,  86 

Micheli,  150 

Moller,  63 

Moreschi,  142 

Morgenroth,  10,  13,  16,  77,  131,  134,  139,  145, 

I5° 

Moro,  43,  48 
Moser,  198 
Much,  92 
Miiller,  94,  95,  161,  167,  169 

Nakayama,  143,  145,  158 

Neisser,  A.,  153 

Neisser,  M.,  128,  130,  142,  149,  150,  171,  193 

Netter,  77 

Neufeld,  177 

Noguchi,  90,  1 68,  170 

Obermeyer,  30,  116,  117 
Oppenheim,  161 
Ostertag,  29 


Otto,  82 

Pane,  199 

Parvu,  154 

Pasteur,  3,  25,  26,  27,  39 

Petit,  54 

Petruschky,  61 

Pfeiffer,  32,  42,  100,  118,  121-128,  130,  147, 

175,  176 
Phisalix,  92 
Pick,  30,  116,  117 
v.  Pirquet,  43,  48,  52,  195 
Plato,  54 
Plaut,  in,  150 
Porges,  no,  in,  167 
Potzl,  167,  169 
Prausnitz,  88 

Rabinowitsch,  145 
Ransom,  80 
Reschad,  66 
Ribbert,  194 
Rietschel,  153 
Romer,  87,  199 
Ropke,  45,  63,  64 
Rosculet,  85 
Rosenau,  80,  195 
Rosenblatt,  147 
Rosenthal,  83,  84 
Roux,  68,  71,  76,  79 
Ruppel,  65,  195,  198 

Sachs,  H.,  82,  90,  142,  167,  171 

Sahli,  58 

Satimbeni,  200 

Salmon,  30 

Salomonsen,  72,  73 

Salus,  42 

Schenck,  54 

Schick,  195 

Schleissner,  157,  198 

Schone,  161 

Schucht,  149,  150 

Schulze,  85,  86 

Schiitze,  113,  150 

Schwarz,  75 

Sclavo,  201 

Seiffert,  54 

Seligmann,  150 

Shiga,  42,  83,  1 06 

Smith,  30 

Smith,  Theob.,  195 

Sobernheim,  201 

Spengler,  57,  66 

Steinberg,  130 

Steinhardt,  195 

Stenitzer,  201 

Stern,  Marg.,  150,  168,  170 

Stern,  128 

Stertz,  150 

Strauss,  13 

Szaboky,  92 

Takaki,  79 
Tallquist,  93 
Tavel,  200,  20 1 
Terni,  200 
Todd,  83,  84 
Topfer,  129,  130 
Toussaint,  30 


INDEX    OF   AUTHORS. 


209 


Trebing,  94 
Tschernogubow,  170 
Tschistowitsch,  108 

Uhlenhuth,  113-117 
Vaillard,  84 

Wassermann,  A.,  2,  3,  36,  57,  59,  79,  82,  105, 
112,  113,  142,  143,  144,  148,  149,  i5°> 
162,  169,  171 

Wechsberg,  85,  128,  130,  193 


Weidanz,  170 

Weigert,  146 

Weil,  33,  39,  143,  J45>  158 

Weinberg,  154,  171 

Widal,  99,  105,  141 

Wolff-Eisner,  48,  192 

Wright,  13,  31,  32,  42,  100,  147,  T76>  l86>  187, 

189,   201 

Yersin,  68,  200 
Zupnik,  80 


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